Methods of treating immunotherapy-related toxicity using a gm-csf antagonist

ABSTRACT

Methods of inhibiting or reducing the incidence or the severity of immunotherapy-related toxicity in a subject, the method comprising a step of administering a recombinant hGM-CSF antagonist to the subject, wherein said administering inhibits or reduces the incidence or the severity of immunotherapy-related toxicity in said subject, are provided. An hGM-CSF antagonist for use in methods of inhibiting or reducing the incidence or the severity of immunotherapy-related toxicity in a subject also are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.62/567,187, filed Oct. 2, 2017, and 62/729,043, filed Sep. 10, 2018,which are hereby incorporated by reference.

FIELD OF THE DISCLOSURE

The disclosure herein provides methods of inhibiting or reducing theincidence and/or the severity of immunotherapy-related toxicity in asubject, the method comprising administering a recombinant GM-CSFantagonist to the subject.

BACKGROUND

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokinesecreted by various cell types including macrophages, T cells, mastcells, natural killer cells, endothelial cells and fibroblasts. GM-CSFstimulates the differentiation of granulocytes and of monocytes.Monocytes, in turn, migrate into tissue and mature into macrophages anddendritic cells. Thus, secretion of GM-CSF leads to a rapid increase inmacrophage numbers. GM-CSF is also involved in the inflammatory responsein the Central Nervous System (CNS) causing influx of blood-derivedmonocytes and macrophages, and the activation of astrocytes andmicroglia. Immuno-related toxicities comprise potentiallylife-threatening immune responses that occur as a result of the highlevels of immune activation occurring from different immunotherapies.Immuno-related toxicity is currently a major complication for theapplication of immunotherapies in cancer patients. It is clear thatthere remains a critical need for methods of preventing and treatingimmuno-related toxicity. An ideal method will minimize the risk of theselife-threatening complications without affecting the efficacy of theimmunotherapy and could potentially even improve the efficacy byallowing, for example, safe increased dosing of immunotherapeuticcompounds and/or an expansion of T cells.

BRIEF SUMMARY OF THE INVENTION

In one aspect, disclosed herein is a method of inhibiting or reducingthe incidence or the severity of immunotherapy-related toxicity in asubject, the method comprising a step of administering a recombinantGM-CSF antagonist to the subject.

In a related aspect, said immunotherapy comprises adoptive celltransfer, administration of monoclonal antibodies, administration ofcytokines, administration of a cancer vaccine, T cell engagingtherapies, or any combination thereof.

In another aspect, adoptive cell transfer comprises administeringchimeric antigen receptor-expressing T-cells (CAR T-cells), T-cellreceptor (TCR) modified T-cells, tumor-infiltrating lymphocytes (TIL),chimeric antigen receptor (CAR)-modified natural killer cells, ordendritic cells, or any combination thereof. In a related aspect, themonoclonal antibody is selected from a group comprising: anti-CD3,anti-CD52, anti-PD1, anti-PD-L1, anti-CTLA4, anti-CD20, anti-BCMAantibodies, bi-specific antibodies, or bispecific T-cell engager (BiTE)antibodies, or any combination thereof. In a related aspect, thecytokines are selected from a group comprising: IFNα, IFNβ, IFNγ, IFNλ,IL-1, IL-2, IL-6, IL-7, IL-15, IL-21, IL-11, IL-12, IL-18, GM-CSF, TNFα,or any combination thereof.

In another aspect, inhibiting or reducing the incidence or the severityof immunotherapy-related toxicity comprises reducing the concentrationof at least one inflammation-associated factor in the serum, tissuefluid, or in the CSF of the subject. In a related aspect, theinflammation-associated factor is selected from a group comprising:C-reactive protein, GM-CSF, IL-1, IL-2, sIL2Rα, IL-5, IL-6, IL-8, IL-10,IP10, IL-15, MCP-1 (AKA CCL2), MIG, MIP1β, IFNγ, CX3CR1, or TNFα, or anycombination thereof. In another aspect, the administration ofrecombinant GM-CSF antagonist does not reduce the efficacy of saidimmunotherapy. In another aspect, the administration of recombinantGM-CSF antagonist increases the efficacy of said immunotherapy. Inanother aspect, administration of recombinant GM-CSF antagonist occursprior to, concurrent with, or following immunotherapy. In a relatedaspect, the recombinant GM-CSF antagonist is co-administered withcorticosteroids, anti-IL-6 antibodies, tocilizumab, anti-IL-1antibodies, cyclosporine, antiepileptic s, benzodiazepines,acetazolamide, hyperventilation therapy, or hyperosmolar therapy, or anycombination thereof.

In another aspect, the immunotherapy-related toxicity comprises a braindisease, damage or malfunction. In a related aspect, the brain disease,damage or malfunction comprises CAR-T cell related neurotoxicity orCAR-T cell related encephalopathy syndrome (CRES). In a related aspect,inhibiting or reducing incidence of a brain disease, damage ormalfunction comprises reducing headaches, delirium, anxiety, tremor,seizure activity, confusion, alterations in wakefulness, hallucinations,dysphasia, ataxia, apraxia, facial nerve palsy, motor weakness,seizures, nonconvulsive EEG seizures, altered levels of consciousness,coma, endothelial activation, vascular leak, intravascular coagulation,or any combination thereof in the subject. In another aspect, theimmunotherapy-related toxicity comprises CAR-T induced Cytokine ReleaseSyndrome (CRS). In a related aspect, inhibiting or reducing incidence ofCRS comprises reducing or inhibiting, without limitation, high fever,myalgia, nausea, hypotension, hypoxia, or shock, or a combinationthereof. In a related aspect, the immunotherapy-related toxicity islife-threatening.

In another aspect, the serum concentration of ANG2 or VWF, or the serumANG2:ANG1 ratio of the subject is reduced. In a related aspect, thesubject has a body temperature above 38° C., an IL-6 serumconcentration>16 pg/ml, or an MCP-1 serum concentration above 1,300pg/ml during the first 36 hours after infusion of said CAR-T cells. In arelated aspect, the subject is predisposed to have said brain disease,damage or malfunction. In a related aspect, the subject has an ANG2:ANG1ratio in serum above 1 prior to the infusion of said CAR-T cells.

In another aspect, the immunotherapy-related toxicity compriseshemophagocytic lymphohistiocytosis (HLH) or macrophage-activationsyndrome (MAS). In a related aspect, inhibiting or reducing incidence ofHLH or MAS comprises increasing survival time and/or time to relapse,reducing macrophage activation, reducing T cell activation, reducing theconcentration of IFNγ in the peripheral circulation, or reducing theconcentration of GM-CSF in the peripheral circulation, or anycombination thereof.

In another aspect, the subject presents with fever, splenomegaly,cytopenias involving two or more lines, hypertriglyceridemia,hypofibrinogenemia, hemophagocytosis, low or absent NK-cell activity,ferritin serum concentration above 500 U/ml, or soluble CD25 serumconcentration above 2400 U/ml, or any combination thereof. In a relatedaspect, the subject is predisposed to acquiring HLH or MAS. In a relatedaspect, the subject carries a mutation in a gene selected from: PRF1,UNC13D, STX11, STXBP2, or RAB27A, or has reduced expression of perforin,or any combination thereof.

In one embodiment, the GM-CSF antagonist is an anti-humanGM-CSF antibody(anti-hGM-CSF antibody). In another embodiment, the anti-GM-CSF antibodyblocks binding of GM-CSF to the alpha subunit of the GM-CSF receptor. Inanother embodiment, the anti-GM-CSF antibody is a polyclonal antibody.In another embodiment, the anti-GM-CSF antibody is a monoclonalantibody. In another embodiment, the anti-hGM-CSF antibody is anantibody fragment that is a Fab, a Fab′, a F(ab′)2, a scFv, or a dAB. Insome embodiments, the monoclonal anti-hGM-CSF antibody, the single-chainFv, and the Fab may be generated in the chicken; chicken IgY are avianequivalents of mammalian IgG antibodies. (Park et al., BiotechnologyLetters (2005) 27:289-295; Finley et al., Appl. Environ. Microbiol., May2006, p. 3343-3349). Chicken IgY antibodies have the followingadvantages: higher avidity, i.e., overall strength of binding between anantibody and an antigen, higher specificity (less cross reactivity withmammalian proteins other than the immunogen); high yield in the eggyolk, and lower background (the structural difference in the Fc regionof IgY and IgG results in less false positive staining). In anotherembodiment, the anti-hGM-CSF antibody may be a camelid, e.g., allama-derived single variable domain on a heavy chain antibodies lackinglight chains (also called sdAbs, VHHs and Nanobodies®); the VHH domain(about 15 kDa) is the smallest known antigen recognition site thatoccurs in mammals having full binding capacity and affinities(equivalent to conventional antibodies). (Garaicoechea et al. (2015)PLoS ONE 10(8): e0133665; Arbabi-Ghahroudi M (2017) Front. Immunol.8:1589; Wu et al., Translational Oncology (2018) 11, 366-373). Inanother embodiment, the antibody fragment is conjugated to polyethyleneglycol. In another embodiment, the anti-GM-CSF antibody has an affinityranging from about 5 pM to about 50 pM. In another embodiment,anti-GM-CSF antibody is a neutralizing antibody. In another embodiment,the anti-GM-CSF antibody is a recombinant or chimeric antibody. Inanother embodiment, the anti-GM-CSF antibody is a human antibody. Inanother embodiment, the anti-GM-CSF antibody comprises a human variableregion. In another embodiment, the anti-GM-CSF antibody comprises anengineered human variable region. In another embodiment the anti-GM-CSFantibody comprises a humanized variable region. In another embodiment,the anti-GM-CSF antibody comprises an engineered human variable region.In another embodiment the anti-GM-CSF antibody comprises a humanizedvariable region.

In one embodiment, the anti-GM-CSF antibody comprises a human lightchain constant region. In another embodiment, the anti-GM-CSF antibodycomprises a human heavy chain constant region. In another embodiment,the human heavy chain constant region is a gamma chain. In anotherembodiment, the anti-GM-CSF antibody binds to the same epitope aschimeric 19/2. In another embodiment, the anti-GM-CSF antibody comprisesthe VH region CDR3 and VL region CDR3 of chimeric 19/2. In anotherembodiment, the anti-GM-CSF antibody comprises the VH region and VLregion CDR1, CDR2, and CDR3 of chimeric 19/2.

In one embodiment, the anti-GM-CSF antibody comprises a heavy chainvariable region that comprises a CDR3 binding specificity determinantRQRFPY or RDRFPY, a J segment, and a V-segment, wherein the J-segmentcomprises at least 95% identity to human JH4 (YFDYWGQGTLVTVSS) and theV-segment comprises at least 90% identity to a human germ line VH1 1-02or VH1 1-03 sequence; or a heavy chain variable region that comprises aCDR3 binding specificity determinant comprising RQRFPY. In anotherembodiment, the J segment comprises YFDYWGQGTLVTVSS. In anotherembodiment, the CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. In anotherembodiment, the heavy chain variable region CDR1 or CDR2 can be a humangermline VH1 sequence; or both the CDR1 and CDR2 can be human germlineVH1. In another embodiment, the antibody comprises a heavy chainvariable region CDR1 or CDR2, or both CDR1 and CDR2, as shown in a V_(H)region set forth in FIG. 1. In another embodiment, the anti-GM-CSFantibody has a V-segment that has a V_(H) V-segment sequence shown inFIG. 1. In another embodiment, the V_(H) that has the sequence of VH#1,VH#2, VH#3, VH#4, or VH#5 set forth in FIG. 1.

In another embodiment, the anti-GM-CSF antibody, e.g., that has a heavychain variable region as described in the paragraph above, comprises alight chain variable region that comprises a CDR3 binding specificitydeterminant comprising the amino acid sequence FNK or FNR.

In another embodiment, the anti-GM-CSF antibody comprises a VL regionthat comprises a CDR3 comprising the amino acid sequence FNK or FNR. Inone embodiment, the anti-GM-CSF antibody comprises a human germline JK4region. In another embodiment, the antibody V_(L) region CDR3 comprisesQQFN(K/R)SPLT. In another embodiment, the anti-GM-CSF antibody comprisesa VL region that comprises a CDR3 comprising QQFNKSPLT. In anotherembodiment, the VL region comprises a CDR1, or a CDR2, or both a CDR1and CDR2, of a V_(L) region shown in FIG. 1. In another embodiment, theV_(L) region comprises a V segment that has at least 95% identity to theVKIIIA27 V-segment sequence as shown in FIG. 1. In another embodiment,the V_(L) region has the sequence of VK#1, VK#2, VK#3, or VK#4 set forthin FIG. 1.

In one embodiment, the anti-GM-CSF antibody has a VH region CDR3 bindingspecificity determinant RQRFPY or RDRFPY and a VL region that has a CDR3comprising QQFNKSPLT. In another embodiment, the anti-GM-CSF antibodyhas a VH region sequence set forth in FIG. 1 and a VL region sequenceset forth in FIG. 1. In another embodiment, the VH region or the VLregion, or both the VH and VL region amino acid sequences comprise amethionine at the N-terminus. In another embodiment, the GM-CSFantagonist is selected from the group comprising of an anti-GM-CSFreceptor antibody or a soluble GM-CSF receptor, a cytochrome b562antibody mimetic, a GM-CSF peptide analog, an adnectin, a lipocalinscaffold antibody mimetic, a calixarene antibody mimetic, and anantibody like binding peptidomimetic.

In one embodiment, disclosed herein is a method of increasing theefficacy of CAR-T immunotherapy in a subject, the method comprising astep of administering a recombinant GM-CSF antagonist to the subject,wherein said administering increases the efficacy of CAR-T immunotherapyin said subject. In another embodiment, said administering a recombinantGM-CSF antagonist occurs prior to, concurrent with, or following saidCAR-T immunotherapy. In another embodiment, said increased efficacycomprises increased CAR-T cell expansion, reduced myeloid-derivedsuppressor cell (MDSC) number that inhibit T-cell function, synergy witha checkpoint inhibitor, or any combination thereof. In anotherembodiment, said increased CAR-T cell expansion comprises at least a 50%increase compared to a control. In another embodiment, said increasedCAR-T cell expansion comprises at least a one quarter log expansioncompared to a control. In another embodiment, said increased cellexpansion comprises at least a one-half log expansion compared to acontrol. In another embodiment, said increased cell expansion comprisesat least a one log expansion compared to a control. In anotherembodiment, said increased cell expansion comprises a greater than onelog expansion compared to a control.

In an embodiment, the GM-CSF antagonist comprises a neutralizingantibody. In another embodiment, the neutralizing antibody is amonoclonal antibody.

In an embodiment, disclosed herein is a method of inhibiting or reducingthe incidence or the severity of CAR-T related toxicity in a subject,the method comprising a step of administering a recombinant GM-CSFantagonist to the subject, wherein said administering inhibits orreduces the incidence or the severity of CAR-T related toxicity in saidsubject. In an embodiment, said CAR-T related toxicity comprisesneurotoxicity, CRS, or a combination thereof. In some embodiments, theCAR-T cell related neurotoxicity is reduced by about 50% compared to areduction in neurotoxicity in a subject treated with CAR-T cells and acontrol antibody. In various embodiments, the recombinant GM-CSFantagonist is a GM-CSF neutralizing antibody in accordance withembodiments described herein.

In another embodiment, said inhibiting or reducing incidence of CRScomprises increasing survival time and/or time to relapse, reducingmacrophage activation, reducing T cell activation, or reducing theconcentration of circulating GM-CSF, or any combination thereof. Inanother embodiment, said subject presents with fever (with or withoutrigors, malaise, fatigue, anorexia, myalgia, arthralgia, nausea,vomiting, headache, skin rash, diarrhea, tachypnea, hypoxemia, hypoxia,shock, cardiovascular tachycardia, widened pulse pressure, hypotension,capillary leak, increased early cardiac output, diminished late cardiacoutput, elevated D-dimer, hypofibrinogenemia with or without bleeding,azotemia, transaminitis, hyperbilirubinemia, mental status changes,confusion, delirium, frank aphasia, hallucinations, tremor, dysmetria,altered gait, seizures, organ failure, or any combination thereof.

In another embodiment, the inhibiting or reducing the incidence or theseverity of CAR-T related toxicity comprises preventing the onset ofCAR-T related toxicity.

In another embodiment, disclosed herein is a method of blocking orreducing GM-CSF expression in a cell, comprising knocking out orsilencing GM-CSF gene expression in a cell. In an embodiment, theblocking or reducing of GM-CSF expression comprises short interferingRNS (siRNA), CRISPR, RNAi, DNA-directed RNA interference (ddRNAi), whichis a gene-silencing technique that uses DNA constructs to activate ananimal cell's endogenous RNA interference (RNAi) pathways, or targetedgenome editing with engineered transcription activator-like effectornucleases (TALENs), i.e., artificial proteins composed of a customizablesequence-specific DNA-binding domain fused to a nuclease that cleavesDNA in a nonsequence-specific manner. (Joung and Sander, Nat Rev MolCell Biol. 2013 January; 14(1): 49-55), which is incorporated herein inits entirety by reference. In an embodiment, the cell is a CAR-T cell.

In one embodiment, the subject is a human.

In one embodiment, disclosed herein is a GM-CSF antagonist for use in amethod of inhibiting or reducing the incidence or severity ofimmunotherapy-related toxicity in a subject, the method comprising astep of administering a recombinant GM-CSF antagonist to the subject. Inone embodiment, disclosed herein is a pharmaceutical compositioncomprising an anti-GM-CSF antagonist.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Provides exemplary V_(H) and V_(L) sequences of anti-GM-CSFantibodies.

FIGS. 2A-2B Binding of GM-CSF to Ab1 (FIG. 2A) or Ab2 (FIG. 2B)determined by surface plasmon resonance analysis at 37° C. (Biacore3000). Ab1 and Ab2 were captured on anti Fab polyclonal antibodiesimmobilized on the Biacore chip. Different concentrations of GM-CSF wereinjected over the surface as indicated. Global fit analysis was carriedout assuming a 1:1 interaction using Scrubber2 software.

FIGS. 3A-3B Binding of Ab1 and Ab2 to glycosylated (FIG. 3A) andnon-glycosylated GM-CSF (FIG. 3B). Binding to glycosylated GM-CSFexpressed from human 293 cells or non-glycosylated GM-CSF expressed inE. coli was determined by ELISA. Representative results from a singleexperiment are shown (exp 1). Two-fold dilutions of Ab1 and Ab2 startingfrom 1500 ng/ml were applied to GM-CSF coated wells. Each pointrepresents mean±standard error for triplicate determinations. Sigmoidalcurve fit was performed using Prism 5.0 Software (Graphpad).

FIGS. 4A-4B Competition ELISA demonstrating binding of Ab1 and Ab2 to ashared epitope. ELISA plates coated with 50 ng/well of recombinantGM-CSF were incubated with various concentrations of antibody (Ab2, Ab1or isotype control antibody) together with 50 nM biotinylated Ab2.Biotinylated antibody binding was assayed using neutravidin-HRPconjugate. Competition for binding to GM-CSF was for 1 hr (FIG. 4A) orfor 18 hrs (FIG. 4B). Each point represents mean±standard error fortriplicate determinations. Sigmoidal curve fit was performed using Prism5.0 Software (Graphpad).

FIG. 5 Inhibition of GM-CSF-induced IL-8 expression. Various amounts ofeach antibody were incubated with 0.5 ng/ml GM-CSF and incubated withU937 cells for 16 hrs. IL-8 secreted into the culture supernatant wasdetermined by ELISA.

FIG. 6 Dose-dependent inhibition of GM-CSF-stimulated CD11b on humangranulocytes by anti-GM-CSF antibody.

FIG. 7 Dose-dependent inhibition of GM-CSF-induced HLA-DR on CD14+human, primary monocytes/macrophages by anti-GM-CSF antibody.

FIG. 8 illustrates the role of GM-CSF (Myeloid Inflammatory Factor) as akey cytokine in CAR-T-related activity and in stimulation of white bloodcell proliferation, which is a characteristic feature in certainleukemias, e.g., acute myeloid leukemia (AML).

FIG. 9 Inhibition of GM-CSF-dependent human TF-1 cell proliferation(human erythroleukemia) by neutralization of human GM-CSF withanti-GM-CSF antibody. KB003 is a recombinant monoclonal antibodydesigned to target and neutralize human GM-CSF. KB002 is a chimeric mAblicensed from Ludwig Institute for Cancer Research

FIG. 10 Depiction of chimeric antigen receptor.

FIGS. 11A-11B CAR-T19 Results in high response rates in relapsedrefractory ALL. Data show historic outcomes in R/R ALL and outcomes inR/R ALL after CAR-T19. (Maude, et al NEJM 2014).

FIGS. 12A-12B Evidence showing significant GM-CSF link to neurotoxicity.GM-CSF levels correlate with serious adverse effects after CAR-T celltherapy. GM-CSF levels precede and modulate other cytokines other thanIL-15. Elevated GM-CSF is clearly associated with ≥grade 3 neurotoxicity(NT). IL-2 is only other cytokine with this association.

FIG. 13 Estimated time course of CRS and NT following CD19 CAR-T celltherapy. Timing of symptom onset and CRS severity depends on theinducing agent, type of cancer, age of patient, and the magnitude ofimmune cell activation. CAR-T related CRS symptom onset typically occursdays to occasionally weeks after the T-cell infusion, coinciding withmaximal T-cell expansion. Similar to CRS associated with mAb therapy,CRS associated with adoptive T-cell therapies has been consistentlyassociated with elevated IFNγ, IL-6, TNFα, IL-1, IL-2, IL-6, GM-CSF,IL-10, IL-8, and IL-5. No clear CAR-T cell dose:response relationshipfor CRS exists, but very high doses of T cells may result in earlieronset of symptoms.

FIG. 14 GM-CSF is a key initiator of CAR-T adverse effects. The figuredepicts the central role of GM-CSF in CRS and NT. Perforin allowsgranzymes to penetrate the tumor cell membrane. CAR-T produced GM-CSFrecruits CCR2+ myeloid cells to the tumor site, which produce CCL2(MCP1). CCL2 positively reinforces its own production by CCR2+ myeloidcell recruitment. IL-1 and IL-6 from myeloid cells form another positivefeedback loop with CAR-T by inducing production of GM-CSF. Phosphatidylserine is exposed as a result of perforin and granzyme cell membranedestruction. Phosphatidyl-serine stimulates myeloid cell production ofCCL2, IL-1, IL-6, and other inflammatory effectors. The final outcome ofthis self-reinforcing feedback loop results in endothelial activation,vascular permeability, and ultimately, CRS and neurotoxicity. Moreover,animal model evidence shows GM-CSF knockout mice show no sign of CRS,but IL-6 knockout mice can still develop CRS. GM-CSF receptor k/o fromCCR2+ myeloid cells abrogates cascade in neuro-inflammation models.(Sentman, et al., J. Immunol.; Coxford, et al. Immunity 2015(43)510-514; Ishii et al., Blood 2016 128:3358; Teachey, et al. CancerDiscov. 2016 Jun. 6(6): 664-679; Lee, et al., Blood 2016 124:2:188;Barrett, et al., Blood 2016: 128-654, each of which is incorporated inits entirety herein by reference.).

FIGS. 15A(i), 15A(ii), 15B-15G GM-CSF CRISPR knockout T-cells exhibitreduced expression of GM-CSF but similar levels of other cytokines anddegranulation. a. Generation of GM-CSF knockout CAR-Ts. (See Example 6).

FIGS. 16A(i), 16A(ii), 16B-16J GM-CSF neutralizing antibody inaccordance with embodiments described herein does not inhibit CAR-Tmediated killing, proliferation, or cytokine production but successfullyneutralizes GM-CSF (See Example 7).

FIGS. 17A-17B Protocol and Results from a Mouse Model of Human CRS.(Example 5).

FIGS. 18A-18C CAR-T efficacy in a xenograft model in combination with aGM-CSF neutralizing antibody in accordance with embodiments describedherein. The GM-CSF neutralizing antibody is shown to not inhibit CAR-Tefficacy in vivo. (See Example 8).

FIG. 19 In vitro and In vivo preclinical data showed a GM-CSFneutralizing antibody in accordance with embodiments described hereindid not impair CAR-T impact on survival. The GM-CSF neutralizingantibody does not impede CAR-T cell function in vivo in the absence ofPBMCs. Survival was similar for CAR-T+control and CAR-T+GM-CSFneutralizing antibody. (See Example 9).

FIGS. 20A-20B In vitro and In vivo preclinical data showed a GM-CSFneutralizing antibody in accordance with embodiments described hereinmay increase CAR-T expansion. The GM-CSF neutralizing antibody mayincrease in vitro CAR-T cancer cell killing. The antibody increasesproliferation of CAR-T cells and may improve efficacy. CAR-Tproliferation increased by the GM-CSF neutralizing antibody in presenceof PBMCs. (It was not affected without PBMCs). The antibody did notinhibit CAR-T degranulation, intracellular GM-CSF production, or IL-2production. (See Example 10).

FIG. 21 CAR-T expansion associated with improved overall response rate.CAR AUC (area under the curve) defined as cumulative levels ofCAR+cells/pt of blood over the first 28 days post CAR-T administration.P values calculated by Wilcoxon rank sum test. (Neelapu, et al ICML 2017Abstract 8). (See Example 11).

FIG. 22 Study protocol for GM-CSF neutralizing antibody in accordancewith embodiments described herein. (See Example 12). CRS and NT to beassessed daily while hospitalized and at clinic visit for first 30 days.Eligible subjects to receive GM-CSF neutralizing antibody on days −1,+1, and +3 of CAR-T treatment. Additional dosing can be contemplatedgoing out to at least day 7. Tumor assessment to be performed atbaseline and months 1, 3, 6, 9, 12, 18, and 24. Blood samples (PBMC andserum) days −5, −1, 0, 1, 3, 5, 7, 9, 11, 13, 21, 28, 90, 180, 270, and360. (See Example 12).

FIGS. 23A-23B. GM-CSF depletion increases CAR-T cell expansion. a.Increased ex-vivo expansion of GM-CSF^(k/o) CAR-T cells compared tocontrol CAR-T cells. B. More robust proliferation after treatment with aGM-CSF neutralizing antibody in accordance with embodiments describedherein. (See Example 13).

FIG. 24. Safety profile of GM-CSF neutralizing antibody in accordancewith embodiments described herein. (See Example 14).

FIGS. 25A-25D GM-CSF neutralizing antibody when added to CAR-T celltherapy demonstrates a 90% reduction in neuroinflammation in mousepreclinical model. FIG. 25A illustrates MRI data in which mice brainsshow clear improvement after administration of CAR-T cells and GM-CSFneutralizing antibody in accordance with embodiments described hereincompared to mice brains showing signs of neurotoxicity(neuroinflammation caused by neurotoxicity) after administration ofCAR-T cells and a control antibody (top row) and compared to untreated(baseline) mice brains (bottom row). FIG. 25B quantitatively illustratesthe percent increase of T2 FLAIR from baseline: there was anapproximately 10% percent increase in brain T2/FLAIR from baseline inmice administered CAR-T and GM-CSF neutralizing antibody in accordancewith embodiments described herein compared to the slightly over 100%increase in mice that had been administered CAR-T cells and controlantibody. As shown in the comparative graph, the about 10% increasepercent in brain T2/FLAIR from baseline in mice administered the CAR-Tand GM-CSF neutralizing antibody is a 90% reduction inneuroinflammation, as measured by brain T2/FLAIR from baseline, comparedto the quantity of neuroinflammation present in mice that received CAR-Tcells and control antibody. FIGS. 25C-25D show that compared tountreated mice (which had 500,000 to 1.5M leukemic cells) and CAR-T pluscontrol antibody (which had between 15,000 and 100,000 leukemic cells),treatment with CAR-T plus GM-CSF neutralizing antibody in accordancewith embodiments described herein led to a significant reduction in thenumber of leukemic cells (decreased to between 500 and 5,000 cells) withimproved overall disease control (See Example 15).

FIGS. 26A-26I show that GM-CSF blockade helps control CART19 toxicitiesand may improve efficacy. FIG. 26A shows CART19 and lenzilumab treatedCART19 are equally effective in survival outcomes in a high tumor burdenNALM6 relapse model compared to UTD (untransduced T cells) (7-8 mice pergroup, n=2). FIGS. 26B-26D show Lenzilumab & anti-mouse GM-CSF antibodycontrolled CRS induced weight loss, neutralized serum human GM-CSF, andreduced expression of serum mouse MCP-1 (monocyte chemoattractantprotein-1) in a primary ALL xenograft CART19 CRS/NT model (3 mice pergroup, *p<0.05). FIG. 26E shows Lenzilumab & anti-mouse GM-CSF antibodyreduced brain inflammation as shown by MRI in a primary ALL xenograftCART19 CRS/NT model (3 mice per group, *p<0.05, **p<0.01). FIGS. 26F-26Gshow CART19+Lenzilumab & anti-mouse GM-CSF antibody treated mice showedreduced CD19+ brain leukemic burden and reduced percentage of brainmacrophages in a primary ALL xenograft CART19 CRS/NT model (3 mice pergroup). FIG. 26H shows CRISPR Cas9 K/O of GM-CSF reduces its expressionvia intracellular staining in CART19 and UTD with NALM6 stimulation.(Representative experiment, n=2) FIG. 26I shows CART19 and GM-CSF K/OCART19 control tumor burden better than UTD, and GM/CSF K/O CART19 cellscontrol tumor burden slightly better than CART19 in a high tumor burdenNALM6 relapse model (6 mice per group, *p<0.05, ****p<0.0001). Errorbars SEM.

DETAILED DESCRIPTION OF THE INVENTION

The present subject matter may be understood more readily by referenceto the following detailed description which forms a part of thisdisclosure. It is to be understood that this disclosure is not limitedto the specific products, methods, conditions or parameters describedand/or shown herein, and that the terminology used herein is for thepurpose of describing particular embodiments by way of example only andis not intended to be limiting of the claimed disclosure.

Immunotherapy-Related Toxicity

A skilled artisan would appreciate that the term “immunotherapy-relatedtoxicity” refers to a spectrum of inflammatory symptoms resulting fromhigh levels of immune activation. Different types of toxicity areassociated with different immunotherapy approaches. In some embodiments,immunotherapy-related toxicity comprises capillary leak syndrome,cardiac disease, respiratory disease, CAR-T-cell-related encephalopathysyndrome (CRES), neurotoxicity, colitis, convulsions, cytokine releasesyndrome (CRS), cytokine storm, decreased left ventricular ejectionfraction, diarrhea, disseminated intravascular coagulation, edema,encephalopathy, exanthema, gastrointestinal bleeding, gastrointestinalperforation, hemophagocytic lymphohistiocytosis (HLH), hepatosis,hypertension, hypophysitis, immune related adverse events,immunohepatitis, immunodeficiencies, ischemia, liver toxicity,macrophage-activation syndrome (MAS), pleural effusions, pericardialeffusions, pneumonitis, polyarthritis, posterior reversibleencephalopathy syndrome (PRES), pulmonary hypertension, thromboembolism,and transaminitis.

While different types of toxicities differ in their pathophysiology andclinical manifestations, they are usually associated with an increase ininflammation-associated factors, such as C-reactive protein, GM-CSF,IL-1, IL-2, sIL-2Rα, IL-5, IL-6, IL-8, IL-10, IP10, IL-15, MCP-1 (AKACCL2), MIG, MIP-1β, IFNγ, CX3CR1, or TNFα. A skilled artisan wouldappreciate that, in some embodiments, the term “inflammation-associatedfactor” comprises molecules, small molecules, peptides, genetranscripts, oligonucleotides, proteins, hormones, and biomarkers thatare affected during inflammation. A skilled artisan would appreciatethat systems affected during inflammation comprises upregulation,downregulation, activation, de-activation, or any kind of molecularmodification. The serum concentration of inflammation-associatedfactors, such as cytokines, can be used as an indicator ofimmunotherapy-related toxicities, and may be expressed as—fold increase,percent (%) increase, net increase or rate of change in cytokine levelsor concentration. The concentration of inflammation-associated factorsin body fluids other than serum can also be used as indicators ofimmunotherapy-related toxicities. In some embodiments, absolute cytokinelevels or concentrations above a certain level or concentration may bean indication of a subject undergoing or about to experience animmunotherapy-related toxicity. In another embodiment, absolute cytokinelevels or concentration at a certain level, for example a level orconcentration normally found in a control subject, may be an indicationof a method for inhibiting or reducing the incidence of animmunotherapy-related toxicity in a subject. A skilled artisan wouldappreciate that the term “cytokine level” may encompass a measure ofconcentration, a measure of fold change, a measure of percent (%)change, or a measure of rate change. Further, the methods for measuringcytokines in blood, cerebrospinal fluid (CSF), saliva, serum, urine, andplasma are well known in the art.

A number of approaches were elaborated to classify the type ofneurotoxicity and manage it accordingly. These classifications are basedon clinical and biological symptoms, as fever, hypotension, hypoxia,organ toxicity, cardiac dysfunction, respiratory dysfunction,gastrointestinal dysfunction, hepatic dysfunction, renal dysfunction,coagulopathy, seizure presence, intracranial pressure, muscle tone,motor performance, ferritin levels, and haemagophagocytosis. Similarly,each type of neurotoxicity can be graded according to its severity.Table 1A (taken from Cellular Therapy Implementation: the MDACCApproach, P. Kebriaei, Feb. 24, 2017) discloses a method for gradingneurotoxicity according to its severity into Grade 1, Grade 2, Grade 3,and Grade 4. However, some of the foregoing symptoms are not typicallyassociated with neurotoxicity. (Lee, et al., Blood 2014; 124:188-195,which is incorporated in its entirety herein by reference.).

TABLE 1A Method for Grading Neurotoxicity - Criteria for Adverse Events(CTCAE) Symptom or sign Grade 1 Grade 2 Grade 3 Grade 4 Level of MildModerate somnolence, Obtundation or stupor Life-threateningconsciousness drowsiness/ limiting instrumental needing urgentsleepiness ADL intervention or mechanical ventilation Orientation/ MildModerate Severe disorientation, Life-threatening Confusiondisorientation/ disorientation, limiting self-care ADL needing urgentconfusion limiting instrumental intervention or ADL mechanicalventilation ADL/ Mild limiting Limiting instrumental Limiting self-careLife-threatening Encephalopathy of ADL ADL ADL needing urgentintervention or mechanical ventilation Speech Dysphasia Dysphasia withSevere receptive or not impairing moderate impairment expressivedysphasia, ability to in ability to impairing ability to communicatecommunicate read, write or spontaneously communicate intelligiblySeizure Brief partial Brief generalized Multiple seizuresLife-threatening; seizure; no seizure despite medical prolonged loss ofintervention repetitive seizures consciousness Incontinent or motorBowel/bladder weakness incontinence; Weakness limiting selfcare ADL,disabling MD Mild (7-9) Moderate (3-6) Severe (1-2), grade 1 Critical(Obtunded; Anderson Cancer and 2 papilledema convulsive status Center(MDACC) with CSF opening epilepticus; motor 10-point pressure (op) <20mmHg weakness, grade 3, Neurotoxicity grade 4 & 5 papilledema, CSF op≥20 mmHg, cerebral edema)

Patients with body temperature above 38.9° C., IL-6 serum concentrationabove 16 pg/ml, or MCP-1 (AKA CCL2) serum concentration above 1,343.5pg/ml in the first 36 hours after immunotherapy infusion had higherprobabilities of developing severe neurotoxicity (Gust, et al. CancerDiscov. 2017 Oct. 12).

CRS is a serious condition and life-threatening adverse effect becauseof abnormal cytokine regulation and thus, severe inflammation. Symptomscan include, without limitation, fever, disordered heartbeat andbreathing, nausea, vomiting, and seizures. CRS can be graded byassessing symptoms and their severities, such as, for example: Grade 1CRS: Fever, constitutional symptoms; Grade 2 CRS: Hypotension—respondsto fluids or one low dose pressor, Hypoxia—responds to <40% O₂, Organtoxicity; grade 2; Grade 3 CRS: Hypotension—requires multiple pressorsor high dose pressors, Hypoxia—requires ≥40% O₂, Organ toxicity—grade 3,grade 4 transaminitis; Grade 4 CRS: Mechanical ventilation, Organtoxicity—grade 4, excluding transaminitis. (Lee, et al., Blood 2014;124:188-195, which is incorporated in its entirety herein byreference.).

CRES can be graded, for example, by combining neurological assessmentwith other parameters as papilloedema, CSF opening pressure, imagingassessment, and the presence of seizures and motor weakness. A methodfor grading CRES is described in Neelapu et al., Nat Rev Clin Oncol.15(1):47-62 (2018) (Epub 2017 Sep. 19), which is incorporated in itsentirety herein by reference. Table 1B (taken from Neelapu et al., NatRev Clin Oncol. 15(1):47-62 (2018)) discloses a method for grading CRESaccording to its severity into Grade 1, Grade 2, Grade 3, and Grade 4.

TABLE 1B Method for grading CRES. In CARTOX-10, a point is assigned foreach of the following tasks performed correctly: orientation to year,month, city, hospital, and President/Prime Minister of country ofresidence (1 point for each); naming three objects (1 point for each);writing a standard sentence; counting backwards from 100 in tens.Symptom or sign Grade 1 Grade 2 Grade 3 Grade 4 Neurological 7-9 (mild3-6 0-2 (severe Patient in critical condition, assessment scoreimpairment) (moderate impairment) and/or (by CARTOX-10) impairment)obtunded and cannot perform assessment of tasks Raised intracranial NANA Stage 1-2 Stage 3-5 papilloedema, or pressure papilloedema, or CSFopening CSF pressure ≥20 mmHg, or opening pressure cerebral oedema <20mmHg Seizures or motor NA NA Partial seizure, or Generalized seizures,or weakness non-convulsive convulsive or seizures on EEG non-convulsivestatus with response to epilepticus, or new benzodiazepine motorweakness

Neurotoxicity, CRS, and CRES manifestations can include encephalopathy,headaches, delirium, anxiety, tremor, seizure activity, confusion,alterations in wakefulness, decreased level of consciousness,hallucinations, dysphasia, aphasia, ataxia, apraxia, facial nerve palsy,motor weakness, seizures, nonconvulsive EEG seizures, cerebral edema,and coma. CRES is associated with elevated concentrations of circulatingcytokines, as C-reactive protein, GM-CSF, IL-1, IL-2, sIL2Rα, IL-5,IL-6, IL-8, IL-10, IP10, IL-15, MCP-1, MIG, MIP1β, IFNγ, CX3CR1, andTNFα.

The cytokine concentration gradient between serum and CSF observed innormal conditions is reduced or lost during CRES. Additionally, CART-cells and high protein concentrations are observed in the CSF ofpatients and is correlated with the severity of the condition. All thisindicates a blood-brain barrier dysfunction following immunotherapy.Increased vascular permeability can be partially explained by increasedconcentrations of ANG2 and increased ANG2:ANG1 ratio in patients withneurotoxicity. While ANG1 induces endothelial cell quiescence, ANG2causes endothelial cell activation and microvascular permeability.Patients with increased endothelial activation before immunotherapy werereported to have higher probability of suffering neurotoxicity (Gust, etal. Cancer Discov. 2017 Oct. 12).

Hemophagocytic lymphohistiocytosis (HLH) comprises severehyperinflammation caused by uncontrolled proliferation of benignlymphocytes and macrophages that secrete high amounts of inflammatorycytokines. In some embodiments, HLH can be classified as one of thecytokine storm syndromes. In some embodiments, HLH occurs after strongimmunologic activation, such as systemic infections, immunodeficiency,malignancies. or immunotherapy. In some embodiments, the term “HLH” maybe used interchangeably with the terms “hemophagocyticlymphohistiocytosis”, “hemophagocytic syndrome”, or “hemophagocyticsyndrome” having all the same qualities and meanings.

Primary HLH comprises a heterogeneous autosomal recessive disorder.Patients with homozygous mutations in one of several genes, exhibit lossof function of proteins involved in cytolytic granule exocytosis. Insome embodiments, HLH can present in infancy with minimal or no trigger.Secondary HLH, or acquired HLH, occurs after strong immunologicactivation, such as that which occurs with systemic infection,immunodeficiency, an underlying malignancy, or immunotherapies. Bothforms of HLH are characterized by an overwhelming activation of normal Tlymphocytes and macrophages, invariably leading to clinical andhaematologic alterations and death in the absence of treatment.

In some embodiments, HLH can be initiated by viral infections, EBV, CMV,parvovirus, HSV, VZV, HHV8, HIV, influenza, hepatitis A, hepatitis B,hepatitis C, bacterial infections, gram-negative rods, Mycoplasmaspecies and Mycobacterium tuberculosis, parasitic infections, Plasmodiumspecies, Leishmania species, Toxoplasma species, fungal infections,Cryptococcal species, Candidal species and Pneumocystis species, amongothers.

Macrophage-activation syndrome (MAS) comprises a condition comprisinguncontrolled activation and proliferation of macrophages, and Tlymphocytes, with a marked increase in circulating cytokine levels, suchas IFNγ, and GM-CSF. MAS is closely related to secondary HLH. MASmanifestations include high fever, hepatosplenomegaly, lymphadenopathy,pancytopenia, liver dysfunction, disseminated intravascular coagulation,hemophagocytosis, hypofibrinogenemia, hyperferritinemia, andhypertriglyceridemia.

CRS comprises a non-antigen-specific immune response similar to thatfound in severe infection. CRS is characterized by any or all of thefollowing symptoms: fever with or without rigors, malaise, fatigue,anorexia, myalgias, arthalgias, nausea, vomiting, headache, skin rash,diarrhea, tachypnea, hypoxemia, hypoxia, shock, cardiovasculartachycardia, widened pulse pressure, hypotension, capillary leak,increased cardiac output (early), potentially diminished cardiac output(late), elevated D-dimer, hypofibrinogenemia with or without bleeding,azotemia, transaminitis, hyperbilirubinemia, headache, mental statuschanges, confusion, delirium, word finding difficulty or frank aphasia,hallucinations, tremor, dysmetria, altered gait, seizures, organfailure, multi-organ failure. Deaths have also been reported. Severe CRShas been reported to occur in up to 60% of patients receiving CAR-T19.

Cytokine storm comprises an immune reaction consisting of a positivefeedback loop between cytokines and white blood cells, with highlyelevated levels of various cytokines. The term “cytokine storm” may beused interchangeably with the terms “cytokine cascade” and“hypercytokinemia” having all the same qualities and meanings. In someembodiments, a cytokine storm is characterized by IL-2 release andlymphoproliferation. Cytokine storm leads to potentiallylife-threatening complications including cardiac dysfunction, adultrespiratory distress syndrome, neurologic toxicity, renal and/or hepaticfailure, and disseminated intravascular coagulation.

As noted, CAR-T cell therapy is currently limited by the risk oflife-threatening neurotoxicity and CRS. Despite active management, allCAR-T responders experience some degree of CRS. Up to 50% of patientstreated with CD19 CAR-T have at least Grade 3 CRS or neurotoxicity.GM-CSF levels and T-cell expansion are the factors most associated withgrade 3 or higher CRS and neurotoxicity.

Reducing or eliminating CRS and neurotoxicity in immunotherapies such asCAR-T is of great value and it is crucial to determine what is drivingor exacerbating the signature CAR-T inflammatory response. Although manycytokines, signaling molecules, and cell types are involved in thispathway, GM-CSF is the one cytokine that appears to be at the center ofthe pathway. Normally undetectable in human serum, it is central to thecyclical positive feedback loop that drives inflammation to the extremesof cytokine storms and endothelial activation. Neurotoxicity andcytokine storms are not the result of a simultaneous release ofcytokines, but rather a cascade of inflammation initiated by GM-CSFresulting in the trafficking and recruitment of myeloid cells to thetumor site. These myeloid cells produce the cytokines observed in CRSand neurotoxicity, perpetuating the inflammatory cascade.

Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF)

As used herein, “Granulocyte Macrophage-Colony Stimulating Factor”(GM-CSF) refers to a small, naturally occurring glycoprotein withinternal disulfide bonds having a molecular weight of approximately 23kDa. In some embodiments, GM-CSF refers to human GM-CSF. In someembodiments, GM-CSF refers to non-human GM-CSF. In humans, it is encodedby a gene located within the cytokine cluster on human chromosome 5. Thesequence of the human gene and protein are known. The protein has anN-terminal signal sequence, and a C-terminal receptor binding domain(Rasko and Gough In: The Cytokine Handbook, A. Thomson, et al, AcademicPress, New York (1994) pages 349-369). Its three-dimensional structureis similar to that of the interleukins, although the amino acidsequences are not similar GM-CSF is produced in response to a number ofinflammatory mediators by mesenchymal cells present in the hemopoieticenvironment and at peripheral sites of inflammation. GM-CSF is able tostimulate the production of neutrophilic granulocytes, macrophages, andmixed granulocyte-macrophage colonies from bone marrow cells and canstimulate the formation of eosinophil colonies from fetal liverprogenitor cells. GM-CSF can also stimulate some functional activitiesin mature granulocytes and macrophages. GM-CSF, a cytokine present inthe bone marrow microenvironment, recruits inflammatory monocyte-deriveddendritic cells, secretes high levels of IL-6 and CCL2/MCP-1, and leadsto a feedback loop, recruiting more monocytes, inflammatory dendriticcells to the inflammation site.

As noted, CRS involves the increase of several cytokines and chemokines,including IFN-γ, IL-6, IL-8, CCL2 (MCP-1), CCL3 (MIP1a), and GM-CSF.(Teachey, D. et al. (June 2016), Cancer Discovery, CD-16-0040; MorganR., et al., (April 2010), Molecular Therapy.). IL-6, one of the keyinflammatory cytokines, is not produced by CAR-T cells. (Barrett, D. etal. (2016), Blood). Instead, it is produced by myeloid cells, which arerecruited to the tumor site. GM-CSF mediates this recruitment, whichinduces chemokine production that activates myeloid cells and causesthem to traffic to the tumor site. Elevated GM-CSF levels serve as botha predictive biomarker for CRS and an indicator of its severity. Morethan a critical component of the inflammation cascade, GM-CSF is the keyinitiator, responsible for both CRS and neurotoxicity. As describedherein, in vivo studies using murine models indicate that geneticsilencing of GM-CSF prevents cytokine storm—while still maintainingCAR-T efficacy. GM-CSF knockout mice have normal levels of INF-γ, IL-6,IL-10, CCL2 (MCP1), CCL3/4 (MIG-1) in vivo and do not develop CRS.(Sentman, M.-L., et al (2016), The Journal of Immunology, 197(12),4674-4685.). GM-CSF knockout CAR-T models recruit fewer NK cells, CD8cells, myeloid cells, and neutrophils to the tumor site in comparison toGM-CSF+CAR-T.

The term “soluble granulocyte macrophage-colony stimulating factorreceptor” (sGM-CSFR) refers to a non-membrane bound receptor that bindsGM-CSF, but does not transduce a signal when bound to the ligand.

As used herein, a “peptide GM-CSF antagonist” refers to a peptide thatinteracts with GM-CSF, or its receptor, to reduce or block (eitherpartially or completely) signal transduction that would otherwise resultfrom the binding of GM-CSF to its cognate receptor expressed on cells.GM-CSF antagonists may act by reducing the amount of GM-CSF ligandavailable to bind the receptor (e.g., antibodies that once bound toGM-CSF increase the clearance rate of GM-CSF) or prevent the ligand frombinding to its receptor either by binding to GM-CSF or the receptor(e.g., neutralizing antibodies). GM-CSF antagonists may also includeother peptide inhibitors, which may include polypeptides that bindGM-CSF or its receptor to partially or completely inhibit signaling. Apeptide GM-CSF antagonist can be, e.g., an antibody; a natural orsynthetic GM-CSF receptor ligand that antagonizes GM-CSF, or otherpolypeptides. An exemplary assay to detect GM-CSF antagonist activity isprovided in Example 1. Typically, a peptide GM-CSF antagonist, such as aneutralizing antibody, has an EC50 of 10 nM or less.

A “purified” GM-CSF antagonist as used herein refers to a GM-CSFantagonist that is substantially or essentially free from componentsthat normally accompany it as found in its native state. For example, aGM-CSF antagonist such as an anti-GM-CSF antibody that is purified fromblood or plasma is substantially free of other blood or plasmacomponents such as other immunoglobulin molecules. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis orhigh-performance liquid chromatography. A protein that is thepredominant species present in a preparation is substantially purified.Typically, “purified” means that the protein is at least 85% pure, morepreferably at least 95% pure, and most preferably at least 99% purerelative to the components with which the protein naturally occurs.

Antibodies

As used herein, an “antibody” refers to a protein functionally definedas a binding protein and structurally defined as comprising an aminoacid sequence that is recognized by one of skill as being derived fromthe framework region of an immunoglobulin-encoding gene of an animalthat produces antibodies. An antibody can consist of one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains, respectively.

The term “antibody” includes antibody fragments that retain bindingspecificity. For example, there are a number of well characterizedantibody fragments. Thus, for example, pepsin digests an antibodyC-terminal to the disulfide linkages in the hinge region to produceF(ab′)2, a dimer of Fab which itself is a light chain joined to VH-CH1by a disulfide bond. The F(ab′)2 may be reduced under mild conditions tobreak the disulfide linkage in the hinge region thereby converting the(Fab′)2 dimer into a Fab′ monomer. The Fab′ monomer is essentially anFab with part of the hinge region (see, Fundamental Immunology, W. E.Paul, ed., Raven Press, N.Y. (1993), for a more detailed description ofother antibody fragments). While various antibody fragments are definedin terms of the digestion of an intact antibody, one of skill willappreciate that fragments can be synthesized de novo either chemicallyor by utilizing recombinant DNA methodology. Thus, the term antibody, asused herein also includes antibody fragments either produced by themodification of whole antibodies or synthesized using recombinant DNAmethodologies.

Antibodies include dimers such as V_(H)—V_(L) dimers, V_(H) dimers, orV_(L) dimers, including single chain antibodies (antibodies that existas a single polypeptide chain), such as single chain Fv antibodies (sFvor scFv), in which a variable heavy and a variable light region arejoined together (directly or through a peptide linker) to form acontinuous polypeptide. The single chain Fv antibody is a covalentlylinked V_(H)-V_(L) heterodimer which may be expressed from a nucleicacid including V_(H)- and V_(L)-encoding sequences either joineddirectly or joined by a peptide-encoding linker (e.g., Huston, et al.Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). While the V_(H) andV_(L) are connected to each as a single polypeptide chain, the V_(H) andV_(L) domains associate non-covalently. Alternatively, the antibody canbe another fragment, such as a disulfide-stabilized Fv (dsFv). Otherfragments can also be generated, including using recombinant techniques.The scFv antibodies and a number of other structures converting thenaturally aggregated, but chemically separated light and heavypolypeptide chains from an antibody V region into a molecule that foldsinto a three-dimensional structure substantially similar to thestructure of an antigen-binding site and are known to those of skill inthe art (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778).In some embodiments, antibodies include those that have been displayedon phage or generated by recombinant technology using vectors where thechains are secreted as soluble proteins, e.g., scFv, Fv, Fab, (Fab′)2 orgenerated by recombinant technology using vectors where the chains aresecreted as soluble proteins. Antibodies for use in the invention canalso include diantibodies and miniantibodies.

Antibodies of the invention also include heavy chain dimers, such asantibodies from camelids. Since the V_(H) region of a heavy chain dimerIgG in a camelid does not have to make hydrophobic interactions with alight chain, the region in the heavy chain that normally contacts alight chain is changed to hydrophilic amino acid residues in a camelid.V_(H) domains of heavy-chain dimer IgGs are called VHH domains.Antibodies for use in the current invention include single domainantibodies (dAbs) and nanobodies (see, e.g., Cortez-Retamozo, et al.,Cancer Res. 64:2853-2857, 2004).

As used herein, “V-region” refers to an antibody variable region domaincomprising the segments of Framework 1, CDR1, Framework 2, CDR2, andFramework 3, including CDR3 and Framework 4, which segments are added tothe V-segment as a consequence of rearrangement of the heavy chain andlight chain V-region genes during B-cell differentiation. A “V-segment”as used herein refers to the region of the V-region (heavy or lightchain) that is encoded by a V gene. The V-segment of the heavy chainvariable region encodes FR1-CDR1-FR2-CDR2 and FR3. For the purposes ofthis invention, the V-segment of the light chain variable region isdefined as extending though FR3 up to CDR3.

As used herein, the term “J-segment” refers to a subsequence of thevariable region encoded comprising a C-terminal portion of a CDR3 andthe FR4. An endogenous J-segment is encoded by an immunoglobulin J-gene.

As used herein, “complementarity-determining region (CDR)” refers to thethree hypervariable regions in each chain that interrupt the four“framework” regions established by the light and heavy chain variableregions. The CDRs are primarily responsible for binding to an epitope ofan antigen. The CDRs of each chain are typically referred to as CDR1,CDR2, and CDR3, numbered sequentially starting from the N-terminus, andare also typically identified by the chain in which the particular CDRis located. Thus, for example, a V_(H) CDR3 is located in the variabledomain of the heavy chain of the antibody in which it is found, whereasa V_(L) CDR1 is the CDR1 from the variable domain of the light chain ofthe antibody in which it is found.

The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three-dimensional space.

The amino acid sequences of the CDRs and framework regions can bedetermined using various well-known definitions in the art, e.g., Kabat,Chothia, international ImMunoGeneTics database (IMGT), and AbM (see,e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structuresfor the hypervariable regions of immunoglobulins. J. Mol. Biol. 196,901-917; Chothia C. et al., 1989, Conformations of immunoglobulinhypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992,structural repertoire of the human VH segments J. Mol. Biol. 227,799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)). Definitions ofantigen combining sites are also described in the following: Ruiz etal., IMGT, the international ImMunoGeneTics database. Nucleic AcidsRes., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the internationalImMunoGeneTics database. Nucleic Acids Res. January 1; 29(1):207-9(2001); MacCallum et al, Antibody-antigen interactions: Contact analysisand binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); andMartin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin,et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al,Immunomethods, 1,126, (1992); and Rees et al, In Sternberg M. J. E.(ed.), Protein Structure Prediction. Oxford University Press, Oxford,141-172 1996).

“Epitope” or “antigenic determinant” refers to a site on an antigen towhich an antibody binds. Epitopes can be formed both from contiguousamino acids or noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed (1996).

The term “binding specificity determinant” or “BSD” as used in thecontext of the current invention refers to the minimum contiguous ornon-contiguous amino acid sequence within a CDR region necessary fordetermining the binding specificity of an antibody. In the currentinvention, the minimum binding specificity determinants reside within aportion or the full-length of the CDR3 sequences of the heavy and lightchains of the antibody.

As used herein, “anti-GM-CSF antibody” or “GM-CSF antibody” are usedinterchangeably to refer to an antibody that binds to GM-CSF andinhibits GM-CSF receptor activity. Such antibodies may be identifiedusing any number of art-recognized assays that assess GM-CSF bindingand/or function. For example, binding assays such as ELISA assays thatmeasure the inhibition of GM-CSF binding to the alpha receptor subunitmay be used. Cell-based assays for GM-CSF receptor signaling, such asassays which determine the rate of proliferation of a GM-CSF-dependentcell line in response to a limiting amount of GM-CSF, are alsoconveniently employed, as are assays that measure amounts of cytokineproduction, e.g., IL-8 production, in response to GM-CSF exposure.

As used herein, “neutralizing antibody” refers to an antibody that bindsto GM-CSF and inhibits signaling by the GM-CSF receptor, or preventsbinding of GM-CSF to its receptor.

As used herein, “human Granulocyte Macrophage-Colony Stimulating Factor”(hGM-CSF) refers to a small naturally occurring glycoprotein withinternal disulfide bonds having a molecular weight of approximately 23kDa; the source and the target of the GM-CSF are human; as such,anti-hGM-CSF antibody, as described in embodiments herein, binds onlyhuman and primate GM-CSF, but not mouse, rat, and other mammalianGM-CSF. The hGM-CSF antibodies, as described in embodiments herein,neutralize human GM-CSF. In some embodiments, the hGM-CSF in humans isencoded by a gene located within the cytokine cluster on humanchromosome 5. The sequences of the human gene and protein are known. Theprotein has an N-terminal signal sequence, and a C-terminal receptorbinding domain (Rasko and Gough In: The Cytokine Handbook, A. Thomson,et al., Academic Press, New York (1994) pages 349-369). Itsthree-dimensional structure is similar to that of the interleukins,although the amino acid sequences are not similar. GM-CSF is produced inresponse to a number of inflammatory mediators present in thehemopoietic environment and at peripheral sites of inflammation. GM-CSFis able to stimulate the production of neutrophilic granulocytes,macrophages, and mixed granulocyte-macrophage colonies from bone marrowcells and can stimulate the formation of eosinophil colonies from fetalliver progenitor cells. GM-CSF can also stimulate some functionalactivities in mature granulocytes and macrophages and inhibits apoptosisof granulocytes and macrophages.

The term “equilibrium dissociation constant” or “affinity” abbreviated(K_(D)), refers to the dissociation rate constant (k_(d), time⁻¹)divided by the association rate constant (k_(a), time⁻¹ M⁻¹).Equilibrium dissociation constants can be measured using any knownmethod in the art. The antibodies of the present invention are highaffinity antibodies. Such antibodies have a monovalent affinity better(less) than about 10 nM, and often better than about 500 pM or betterthan about 50 pM as determined by surface plasmon resonance analysisperformed at 37° C. Thus, in some embodiments, the antibodies of theinvention have an affinity (as measured using surface plasmonresonance), of less than 50 pM, typically less than about 25 pM, or evenless than 10 pM.

In some embodiments, an anti-GM-CSF antibody of the invention has a slowdissociation rate with a dissociation rate constant (kd) determined bysurface plasmon resonance analysis at 37° C. for the monovalentinteraction with GM-CSF less than approximately 10⁻⁴ s⁻¹, preferablyless than 5×10⁻⁵ s⁻¹ and most preferably less than 10⁻⁵ s⁻¹.

As used herein, “chimeric antibody” refers to an immunoglobulin moleculein which (a) the constant region, or a portion thereof, is altered,replaced or exchanged so that the antigen binding site (variable region)is linked to a constant region of a different or altered class, effectorfunction and/or species, or an entirely different molecule that confersnew properties to the chimeric antibody, e.g., an enzyme, toxin,hormone, growth factor, drug, etc.; or (b) the variable region, or aportion thereof, is altered, replaced or exchanged with a variableregion, or portion thereof, having a different or altered antigenspecificity; or with corresponding sequences from another species orfrom another antibody class or subclass.

As used herein, “humanized antibody” refers to an immunoglobulinmolecule in CDRs from a donor antibody are grafted onto human frameworksequences. Humanized antibodies may also comprise residues of donororigin in the framework sequences. The humanized antibody can alsocomprise at least a portion of a human immunoglobulin constant region.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. Humanization can be performed using methods known in the art(e.g., Jones et al., Nature 321:522-525; 1986; Riechmann et al., Nature332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988);Presta, Curr. Op. Struct. Biol. 2:593-596, 1992; U.S. Pat. No.4,816,567), including techniques such as “superhumanizing” antibodies(Tan et al., J. Immunol. 169: 1119, 2002) and “resurfacing” (e.g.,Staelens et al., Mol. Immunol. 43: 1243, 2006; and Roguska et al., Proc.Natl. Acad. Sci USA 91: 969, 1994).

A “HUMANEERED™” antibody in the context of this invention refers to anengineered human antibody having a binding specificity of a referenceantibody. An engineered human antibody for use in this invention has animmunoglobulin molecule that contains minimal sequence derived from adonor immunoglobulin. In some embodiments, the engineered human antibodymay retain only the minimal essential binding specificity determinantfrom the CDR3 regions of a reference antibody. Typically, an engineeredhuman antibody is engineered by joining a DNA sequence encoding abinding specificity determinant (BSD) from the CDR3 region of the heavychain of the reference antibody to human V_(H) segment sequence and alight chain CDR3 BSD from the reference antibody to a human V_(L)segment sequence. A “BSD” refers to a CDR3-FR4 region, or a portion ofthis region that mediates binding specificity. A binding specificitydeterminant therefore can be a CDR3-FR4, a CDR3, a minimal essentialbinding specificity determinant of a CDR3 (which refers to any regionsmaller than the CDR3 that confers binding specificity when present inthe V region of an antibody), the D segment (with regard to a heavychain region), or other regions of CDR3-FR4 that confer the bindingspecificity of a reference antibody. Methods for engineering humanantibodies are provided in US patent application publication no.20050255552 and US patent application publication no. 20060134098.

The term “human antibody” as used herein refers to an antibody that issubstantially human, i.e., has FR regions, and often CDR regions, from ahuman immune system. Accordingly, the term includes humanized andhumaneered antibodies as well as antibodies isolated from micereconstituted with a human immune system and antibodies isolated fromdisplay libraries.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not normally found in the same relationship toeach other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid.Similarly, a heterologous protein will often refer to two or moresubsequences that are not found in the same relationship to each otherin nature.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under-expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus, an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid.

The phrase “specifically (or selectively) binds” to an antibody or is“specifically (or selectively) immunoreactive with”, refers to a bindingreaction where the antibody binds to the antigen of interest. In thecontext of this invention, the antibody typically binds to the antigen,e.g., GM-CSF, with an affinity of 500 nM or less, and has an affinity of5000 nM or greater, for other antigens.

The terms “identical” or percent “identity,” in the context of two ormore polypeptide (or nucleic acid) sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues (or nucleotides) that are the same(i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specifiedregion, when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site). Such sequences are then said to be “substantiallyidentical.” “Substantially identical” sequences also includes sequencesthat have deletions and/or additions, as well as those that havesubstitutions, as well as naturally occurring, e.g., polymorphic orallelic variants, and man-made variants. As described below, thepreferred algorithms can account for gaps and the like. Preferably,protein sequence identity exists over a region that is at least about 25amino acids in length, or more preferably over a region that is 50-100amino acids=in length, or over the length of a protein.

A “comparison window”, as used herein, includes reference to a segmentof one of the number of contiguous positions selected from the groupconsisting typically of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

An indication that two polypeptides are substantially identical is thatthe first polypeptide is immunologically cross reactive with theantibodies raised against the second polypeptide. Thus, a polypeptide istypically substantially identical to a second polypeptide, e.g., wherethe two peptides differ only by conservative substitutions.

Preferred examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity include the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990). BLAST and BLAST 2.0 are used, with the parameters describedherein, to determine percent sequence identity for the nucleic acids andproteins of the invention. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc.Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis orhigh-performance liquid chromatography. A protein that is thepredominant species present in a preparation is substantially purified.The term “purified” in some embodiments denotes that a protein givesrise to essentially one band in an electrophoretic gel. Preferably, itmeans that the protein is at least 85% pure, more preferably at least95% pure, and most preferably at least 99% pure.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidswhich encode identical or essentially identical amino acid sequences, orwhere the nucleic acid does not encode an amino acid sequence, toessentially identical or associated, e.g., naturally contiguous,sequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode most proteins. Forinstance, the codons GCA, GCC, GCG and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to another of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes silent variations of thenucleic acid. One of skill will recognize that in certain contexts eachcodon in a nucleic acid (except AUG, which is ordinarily the only codonfor methionine, and TGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Accordingly, often silent variations of a nucleic acid which encodes apolypeptide is implicit in a described sequence with respect to theexpression product, but not with respect to actual probe sequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables and substitution matrices such asBLOSUM providing functionally similar amino acids are well known in theart. Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention. Typical conservative substitutions for one anotherinclude: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)).

Methods for Preventing or Treating an Immunotherapy-Related Toxicity

In some embodiments, disclosed herein are methods of inhibitingimmunotherapy-related toxicity in a subject. In some embodiments, hereinare methods of reducing the incidence of immunotherapy-related toxicityin a subject. In some embodiments, disclosed herein are methods ofneutralizing hGM-CSF. In some embodiment, the methods comprise a step ofadministering a recombinant hGM-CSF antagonist to the subject. In someembodiments, the method comprises hGM-CSF gene silencing. In someembodiments, the method comprises hGM-CSF gene knockout. Methods of genesilencing and gene knockout are well known to those of ordinary skill inthe art, and may include, without limitation, RNA interference (RNAi),CRISPR, short interfering RNS (siRNA), DNA-directed RNA interference(ddRNAi), targeted genome editing with engineered transcriptionactivator-like effector nucleases (TALENs) or other suitable techniques.

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises reducing immuneactivation. In some embodiments, inhibiting or reducing the incidence orthe severity of immunotherapy-related toxicity comprises amelioratingcapillary leak syndrome. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesameliorating a cardiac dysfunction. In some embodiments, inhibiting orreducing the incidence or the severity of immunotherapy-related toxicitycomprises ameliorating encephalopathy. In some embodiments, inhibitingor reducing the incidence or the severity of immunotherapy-relatedtoxicity comprises alleviating colitis. In some embodiments, inhibitingor reducing the incidence or the severity of immunotherapy-relatedtoxicity comprises inhibiting convulsions. In some embodiments,inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises ameliorating CRS. In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises ameliorating neurotoxicity. Invarious embodiments, the CAR-T cell related neurotoxicity in a subjectis reduced by about 90% compared to a reduction in neurotoxicity in asubject treated with CAR-T cells and a control antibody. In certainembodiments, the recombinant GM-CSF antagonist is an antibody, inparticular, a GM-CSF neutralizing antibody in accordance withembodiments described herein, including Example 15.

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises reducing cytokinestorm symptoms. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesincreasing impaired left ventricular ejection fraction. In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises ameliorating diarrhea. In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises ameliorating disseminatedintravascular coagulation.

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises reducing edema. Insome embodiments, inhibiting or reducing the incidence or the severityof immunotherapy-related toxicity comprises alleviating exanthema. Insome embodiments, inhibiting or reducing the incidence or the severityof immunotherapy-related toxicity comprises reducing gastrointestinalbleeding. In some embodiments, inhibiting or reducing the incidence orthe severity of immunotherapy-related toxicity comprises treating agastrointestinal perforation. In some embodiments, inhibiting orreducing the incidence or the severity of immunotherapy-related toxicitycomprises treating hemophagocytic lymphohistiocytosis (HLH). In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises treating hepatosis. In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises reducing hypotension. In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises reducing hypophysitis.

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises inhibiting immunerelated adverse events. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesreducing immunohepatitis. In some embodiments, inhibiting or reducingthe incidence or the severity of immunotherapy-related toxicitycomprises reducing immunodeficiencies. In some embodiments, inhibitingor reducing the incidence or the severity of immunotherapy-relatedtoxicity comprises treating ischemia. In some embodiments, inhibiting orreducing the incidence or the severity of immunotherapy-related toxicitycomprises reducing liver toxicity. In some embodiments, inhibiting orreducing the incidence or the severity of immunotherapy-related toxicitycomprises treating macrophage-activation syndrome (MAS). In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises reducing neurotoxicitysymptoms.

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises reducing pleuraleffusions. In some embodiments, inhibiting or reducing the incidence orthe severity of immunotherapy-related toxicity comprises reducingpericardial effusions. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesreducing pneumonitis.

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises reducingpolyarthritis. In some embodiments, inhibiting or reducing the incidenceor the severity of immunotherapy-related toxicity comprises treatingposterior reversible encephalopathy syndrome (PRES). In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises reducing pulmonaryhypertension. In some embodiments, inhibiting or reducing the incidenceor the severity of immunotherapy-related toxicity comprises treatingthromboembolism. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesreducing transaminitis. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesreducing a patient's CRES, neurotoxicity (NT), and/or cytokine releasesyndrome (CRS) grade. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesimproving a patient's CARTOX-10 score.

In some embodiments, the immunotherapy is an activation immunotherapy.In some embodiments, immunotherapy is provided as a cancer treatment. Insome embodiments, immunotherapy comprises adoptive cell transfer.

In some embodiments, adoptive cell transfer comprises administration ofa chimeric antigen receptor-expressing T-cell (CAR T-cell). A skilledartisan would appreciate that chimeric antigen receptors (CARs) are atype of antigen-targeted receptor composed of intracellular T-cellsignaling domains fused to extracellular tumor-binding moieties, mostcommonly single-chain variable fragments (scFvs) from monoclonalantibodies. CARs directly recognize cell surface antigens, independentof MHC-mediated presentation, permitting the use of a single receptorconstruct specific for any given antigen in all patients. Initial CARsfused antigen-recognition domains to the CD3ζ activation chain of theT-cell receptor (TCR) complex. While these first-generation CARs inducedT-cell effector function in vitro, they were largely limited by poorantitumor efficacy in vivo. Subsequent CAR iterations have includedsecondary costimulatory signals in tandem with CD3ζ, includingintracellular domains from CD28 or a variety of TNF receptor familymolecules such as 4-1BB (CD137) and OX40 (CD134). Further, thirdgeneration receptors include two costimulatory signals in addition toCD3ζ, most commonly from CD28 and 4-1BB. Second and third generationCARs dramatically improve antitumor efficacy, in some cases inducingcomplete remissions in patients with advanced cancer. In one embodiment,a CAR T-cell is an immunoresponsive cell modified to express CARs, whichis activated when CARs bind to its antigen.

In one embodiment, a CAR T-cell is an immunoresponsive cell comprisingan antigen receptor, which is activated when its receptor binds to itsantigen. In one embodiment, the CAR T-cells used in the compositions andmethods as disclosed herein are first generation CAR T-cells. In anotherembodiment, the CAR T-cells used in the compositions and methods asdisclosed herein are second generation CAR T-cells. In anotherembodiment, the CAR T-cells used in the compositions and methods asdisclosed herein are third generation CAR T-cells. In anotherembodiment, the CAR T-cells used in the compositions and methods asdisclosed herein are fourth generation CAR T-cells.

In some embodiments, adoptive cell transfer comprises administeringT-cell receptor (TCR) modified T-cells. A skilled artisan wouldappreciate that TCR modified T-cells are manufactured by isolatingT-cells from tumor tissue and isolating their TCRα and TCRβ chains.These TCRα and TCRβ are later cloned and transfected to T cells isolatedfrom peripheral blood, which then express TCRα and TCRβ from T-cellsrecognizing the tumor.

In some embodiments, adoptive cell transfer comprises administeringtumor infiltrating lymphocytes (TIL). In some embodiments, adoptive celltransfer comprises administering chimeric antigen receptor(CAR)-modified NK cells. A skilled artisan would appreciate thatCAR-modified NK cells comprise NK cells isolated from the patient orcommercially available NK engineered to express a CAR that recognizes atumor-specific protein.

In some embodiments, adoptive cell transfer comprises administeringdendritic cells.

In some embodiments, immunotherapy comprises administering monoclonalantibodies. In some embodiments, monoclonal antibodies attach tospecific proteins on cancer cells, thus flagging the cells for theimmune system finding and destroying them. In some embodiments,monoclonal antibodies work by inhibiting immune checkpoints, thushindering the inhibition of the immune system by cancer cells. In someembodiments, monoclonal antibodies improve utility of CAR-T to synergizewith checkpoint inhibitors.

In some embodiments, the antibody targets a protein selected from thegroup comprising: SAC, 5T4, activin receptor-like kinase 1, AGS-22M6,alpha-fetoprotein, angiopoietin 2, angiopoietin 3, B7-H3, BAFF, BCMA,C242 antigen, CA-125, carbonic anhydrase 9, CCR4, CD125, CD152, CD184,CD19, CD2, CD20, CD200, CD22, CD221, CD23, CD25, CD27, CD274, CD276,CD28, CD3, CD30, CD33, CD37, CD38, CD4, CD40, CD41, CD44 v6, CD49b, CD5,CD51, CD52, CD54, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CFD, CGRP,ch4D5, CLDN18.2, clumping factor A, CSF1R, CSF2, CTGF, CTLA-4, DLL3,DLL4, DPP4, DRS, EGFL7, EGFR, endoglin, EpCAM, ephrin receptor A3,episialin, ERBB3 (HER3), FAP, FGF 23, fibrin II, beta chain, fibronectinextra domain-B, folate hydrolase, folate receptor, Frizzled receptor,GCGR, GD2 ganglioside, GD3 ganglioside, GDF-8, glypican 3, GM-CSF,GM-CSF receptor α-chain, GPNMB, GUCY2C, HER1, HER2/neu, HGF, HHGFR,histone complex, human scatter factor receptor kinase, human TNF, ICOSL,IFN-α, IGF1, IGF2, IGHE, IL-17A, IL-13, IL1A, IL-2, IL-6, IL-6 receptor,IL-8, IL-9, ILGF2, integrin α4, integrin α5β1, integrin α7 β7, integrinαvβ3, IP10, KIR2D, KLRC1, Lewis-Y antigen, MAGE-A, MCP-1, mesothelin,MIF, MIG, MIP1β, MS4A1, MSLN, MUC1, mucin CanAg, N-glycolylneuraminicacid, NOGO-A, Notch 1, Notch receptor, NRP1, OX-40, PD-1, PDCD1, PDGF-Rα, phosphate-sodium co-transporter, phosphatidylserine, platelet-derivedgrowth factor receptor beta, prostatic carcinoma cells, RHD, RON, RTN4,SDC1, sIL2Rα, SLAMF7, SOST, sphingosine-1-phosphate, Staphylococcusaureus, STEAP1, TAG-72, T-cell receptor, TEM1, tenascin C, TFPI, TGFbeta 1, TGF beta 2, TGF-β, TNFR superfamily member 4, TNF-α, TRAIL-R1,TRAIL-R2, TRP-1, TRP-2, TSLP, tumor antigen CTAA16.88, tumor specificglycosylation of MUC1, tumor-associated calcium signal transducer 2,TWEAK receptor, TYRP1(glycoprotein 75), VEGFA, VEGFR-1, VEGFR2,vimentin, and VWF.

In some embodiments, the antibody is a bi-specific antibody. In someembodiments, the antibody is a bispecific T-cell engager (BiTE)antibody. In some embodiments, the antibody is selected from a groupcomprising: ipilimumab, nivolumab, pembrolizumab, atezolizumab,avelumab, durvalumab, rituximab, TGN1412, alemtuzumab, OKT3 or anycombination thereof.

In some embodiments, immunotherapy comprises administering cytokines. Askilled artisan would appreciate that cytokines can be administered inorder to enhance the immune system to attack the tumor by increasing itsrecognition and killing by immune cytotoxic cells. In some embodiments,the cytokine is selected from a group comprising: IFNα, IFNβ, IFNγ,IFNλ, IL-1, IL-2, IL-6, IL-7, IL-15, IL-21, IL-11, IL-12, IL-18, GM-CSF,TNFα, or any combination thereof.

In some embodiments, immunotherapy comprises administering immunecheckpoint inhibitors. A skilled artisan would appreciate that immunecheckpoints are membranal proteins that keep T cells from attacking thecells that express it. Immune checkpoints are often expressed by cancercells, thus preventing T cells from attacking them. In some embodiments,checkpoint proteins comprise PD-1/PD-L1 and CTLA-4/B7-1/B7-2. Blockingcheckpoint proteins was shown to disengage the inhibition of T cells toattack and kill cancer cells. In some embodiments, checkpoint inhibitorsare selected from a group comprising molecules blocking CTLA-4, PD-1, orPD-L1. In some embodiments, the checkpoint inhibitors are antibodies orparts thereof.

In some embodiments, immunotherapy comprises administeringpolysaccharides. A skilled artisan would appreciate that certainpolysaccharides found in mushroom enhance the immune system and itsanti-cancer properties. In some embodiments polysaccharides arebeta-glucans or lentinan.

In some embodiments, immunotherapy comprises administering or a cancervaccine. A skilled artisan would appreciate that a cancer vaccineexposes the immune system to a cancer-specific antigen and an adjuvant.In some embodiments, the cancer vaccine is selected from a groupcomprising: sipuleucel-T, GVAX, ADXS11-001, ADXS31-001, ADXS31-164,ALVAC-CEA vaccine, AC Vaccine, talimogene laherparepvec, BiovaxlD,Prostvac, CDX110, CDX1307, CDX1401, CimaVax-EGF, CV9104, DNDN, NeuVax,Ae-37, GRNVAC, tarmogens, GI-4000, GI-6207, GI-6301, ImPACT Therapy,IMA901, hepcortespenlisimut-L, Stimuvax, DCVax-L, DCVax-Direct, DCVaxProstate, CBLI, Cvac, RGSH4K, SCIB1, NCT01758328, and PVX-410.

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises decreasing theconcentration of at least one inflammation-associated factor in a bodyfluid. In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises decreasing theconcentration of at least one inflammation-associated factor in theserum. In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises decreasing theconcentration of at least one inflammation-associated factor in thecerebrospinal fluid (CSF). In some embodiments, disclosed herein aremethods for decreasing the concentration of at least oneinflammation-associated factor in serum. In some embodiments, disclosedherein are methods for decreasing the concentration of at least oneinflammation-associated factor in a tissue fluid. In some embodiments,disclosed herein are methods for decreasing the concentration of atleast one inflammation-associated factor in CSF. In some embodiments,the concentration of at least one inflammation-associated factor inserum is decreased. In some embodiments, the concentration of at leastone inflammation-associated factor in a tissue fluid is decreased. Insome embodiments, the concentration of at least oneinflammation-associated factor in CSF is decreased. A skilled artisanwould appreciate that decreasing the concentration of aninflammation-associated factor comprises decreasing or inhibiting theproduction of said inflammation-associated factor in a subject, orinhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity in a subject. In another embodiment,decreasing or inhibiting the production of an inflammation-associatedfactor comprises treating immunotherapy-related toxicity. In anotherembodiment, decreasing or inhibiting the production of aninflammation-associated factor comprises preventingimmunotherapy-related toxicity. In another embodiment, decreasing orinhibiting the production of an inflammation-associated factor levelscomprises alleviating immunotherapy-related toxicity. In anotherembodiment, decreasing or inhibiting the production of aninflammation-associated factor comprises amelioratingimmunotherapy-related toxicity.

In some embodiments, the inflammation-associated factor is a cytokine.In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises decreasing theconcentration of at least one cytokine in the serum. In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises decreasing the concentration ofat least one cytokine in the CSF.

In some embodiments, the cytokine is hGM-CSF. In some embodiments, thecytokine is interleukin (IL)-1β. In some embodiments, the cytokine isIL-2. In some embodiments, the cytokine is sIL2Rα. In some embodiments,the cytokine is IL-5. In some embodiments, the cytokine is IL-6. In someembodiments, the cytokine is IL-8. In some embodiments, the cytokine isIL-10. In some embodiments, the cytokine is IP10. In some embodiments,the cytokine is IL-13. In some embodiments, the cytokine is IL-15. Insome embodiments, the cytokine is tumor necrosis factor α (TNFα). Insome embodiments, the cytokine is interferon γ (IFNγ). In someembodiments, the cytokine is monokine induced by gamma interferon (MIG).In some embodiments, the cytokine is macrophage inflammatory protein(MIP) 1β. In some embodiments, the cytokine is C-reactive protein. Insome embodiments, decreasing or inhibiting the production of cytokinelevels comprises decreasing or inhibiting the production of onecytokine. In some embodiments, decreasing or inhibiting the productionof cytokine levels comprises decreasing or inhibiting the production ofat least one cytokine. In some embodiments, decreasing or inhibiting theproduction of cytokine levels comprises decreasing or inhibiting theproduction of a number of cytokines.

In one embodiment, the methods disclosed herein do not affect theefficacy of the immunotherapy. In another embodiment, the methodsdisclosed herein reduce the efficacy of the immunotherapy by less thanabout 5%. In another embodiment, the methods disclosed herein reduce theefficacy of the immunotherapy by less than about 10%. In anotherembodiment, the methods disclosed herein reduce the efficacy of theimmunotherapy by less than about 15%. In another embodiment, the methodsdisclosed herein reduce the efficacy of the immunotherapy by less thanabout 20%. In another embodiment, the methods disclosed herein reducethe efficacy of the immunotherapy by less than about 50%.

In one embodiment, the methods described herein increase the efficacy ofthe immunotherapy. In one embodiment, increasing the efficacy allows forimprovement of the clinical management, patient outcomes, andtherapeutic index of the immunotherapy. In another embodiment, theincreased efficacy enables administration of higher immunotherapy doses.In another embodiment, the increased efficacy reduces hospitalizationstay and additional treatments and monitoring. In an embodiment, theimmunotherapy comprises CAR-T.

Any appropriate method of quantifying cytotoxicity can be used todetermine whether the immunotherapy efficacy remains substantiallyunchanged. For example, cytotoxicity can be quantified using a cellculture-based assay such as the cytotoxic assays described in theExamples. Cytotoxicity assays can employ dyes that preferentially stainthe DNA of dead cells. In other cases, fluorescent and luminescentassays that measure the relative number of live and dead cells in a cellpopulation can be used. For such assays, protease activities serve asmarkers for cell viability and cell toxicity, and a labeled cellpermeable peptide generates fluorescent signals that are proportional tothe number of viable cells in the sample. In another embodiment, ameasure of cytotoxicity may be qualitative. In another embodiment, ameasure of cytotoxicity may be quantitative.

In an embodiment, said increased efficacy comprises increased CAR-T cellexpansion, reduced myeloid-derived suppressor cells (MDSC) that inhibitT-cell function, synergy with a checkpoint inhibitor, or any combinationthereof. In another embodiment, said increased CAR-T cell expansioncomprises at least a 50% increase compared to a control. In anotherembodiment, said increased CAR-T cell expansion comprises at least a onequarter log expansion compared to a control. In another embodiment, saidincreased cell expansion comprises at least a one-half log expansioncompared to a control. In another embodiment, said increased cellexpansion comprises at least a one log expansion compared to a control.In another embodiment, said increased cell expansion comprises a greaterthan one log expansion compared to a control.

In one embodiment, immunotherapy-related toxicity appears between 2 daysto 4 weeks after administration of immunotherapy. In one embodiment,immunotherapy-related toxicity appears between 0 to 2 days afteradministration of immunotherapy. In some embodiments, the hGM-CSFantagonist is administered to subjects at the same time as immunotherapyas prophylaxis. In another embodiment, the hGM-CSF antagonist isadministered to subjects 0-2 days after administration of immunotherapy.In another embodiment, the hGM-CSF antagonist is administered tosubjects 2-3 days after administration of immunotherapy. In anotherembodiment, the hGM-CSF antagonist is administered to subjects 7 daysafter administration of immunotherapy. In another embodiment, thehGM-CSF antagonist is administered to subjects 10 days afteradministration of immunotherapy. In another embodiment, the hGM-CSFantagonist is administered to subjects 14 days after administration ofimmunotherapy. In another embodiment, the hGM-CSF antagonist isadministered to subjects 2-14 days after administration ofimmunotherapy.

In another embodiment, the hGM-CSF antagonist is administered tosubjects 2-3 hours after administration of immunotherapy. In anotherembodiment, the hGM-CSF antagonist is administered to subjects 7 hoursafter administration of immunotherapy. In another embodiment, thehGM-CSF antagonist is administered to subjects 10 hours afteradministration of immunotherapy. In another embodiment, the GM-CSFantagonist is administered to subjects 14 hours after administration ofimmunotherapy. In another embodiment, the hGM-CSF antagonist isadministered to subjects 2-14 hours after administration ofimmunotherapy.

In an alternative embodiment, the hGM-CSF antagonist is administered tosubjects prior to immunotherapy as prophylaxis. In another embodiment,the hGM-CSF antagonist is administered to subjects 1 day beforeadministration of immunotherapy. In another embodiment, the hGM-CSFantagonist is administered to subjects 2-3 days before administration ofimmunotherapy. In another embodiment, the hGM-CSF antagonist isadministered to subjects 7 days before administration of immunotherapy.In another embodiment, the hGM-CSF antagonist is administered tosubjects 10 days before administration of immunotherapy. In anotherembodiment, the hGM-CSF antagonist is administered to subjects 14 daysbefore administration of immunotherapy. In another embodiment, thehGM-CSF antagonist is administered to subjects 2-14 days beforeadministration of immunotherapy.

In another embodiment, the hGM-CSF antagonist is administered tosubjects 2-3 hours before administration of immunotherapy. In anotherembodiment, the hGM-CSF antagonist is administered to subjects 7 hoursbefore administration of immunotherapy. In another embodiment, thehGM-CSF antagonist is administered to subjects 10 hours beforeadministration of immunotherapy. In another embodiment, the hGM-CSFantagonist is administered to subjects 14 hours before administration ofimmunotherapy. In another embodiment, the hGM-CSF antagonist isadministered to subjects 2-14 hours before administration ofimmunotherapy.

In another embodiment, the hGM-CSF antagonist may be administeredtherapeutically, once immunotherapy-related toxicity has occurred. Inone embodiment, the hGM-CSF antagonist may be administered oncepathophysiological processes leading up to or attesting to the beginningof immunotherapy-related toxicity are detected. In one embodiment, thehGM-CSF antagonist can terminate the pathophysiological processes andavoid its sequelae. In some embodiments, the pathophysiologicalprocesses comprise at least one of the following: increased cytokineconcentrations in serum, increased cytokine concentrations in CSF,increased C-reactive protein (CRP) in serum, increased ferritin in theserum, increased IL-6 in serum, endothelial activation, disseminatedintravascular coagulation (DIC), increased ANG2 serum concentration,increased ANG2:ANG1 ratio in serum, CAR T-cell presence in CSF,increased Von Willebrand factor (VWF) serum concentration,blood-brain-barrier (BBB) leakage, or any combination thereof.

In another embodiment, the hGM-CSF antagonist may be administeredtherapeutically, at multiple time points. In another embodiment,administration of the hGM-CSF antagonist is at least at two time points.In another embodiment, administration of the hGM-CSF antagonist is atleast at three time points.

In one embodiment, the hGM-CSF antagonist is administered once. Inanother embodiment, the hGM-CSF antagonist is administered twice. Inanother embodiment, the hGM-CSF antagonist is administered three times.In another embodiment, the hGM-CSF antagonist is administered fourtimes. In another embodiment, the hGM-CSF antagonist is administered atleast four times. In another embodiment, the hGM-CSF antagonist isadministered more than four times.

A skilled artisan would appreciate that immunotherapy-related toxicityis managed by different treatments. In some embodiments, the hGM-CSFantagonist is co-administered with other treatments. In someembodiments, other treatments are selected from a group comprising:cytokine-directed therapy, anti-IL-6 therapy, cortico steroids,tocilizumab, siltuximab, low-dose vasopressors, inotropic agents,supplemental oxygen, diuresis, thoracentesis, antiepileptics,benzodiazepines, levetiracetam, phenobarbital, hyperventilation,hyperosmolar therapy, and standard therapies for specific organtoxicities.

In some embodiments, immunotherapy-related toxicity comprises a braindisease, damage or malfunction. In some embodiments,immunotherapy-related toxicity comprises CAR T-cell relatedneurotoxicity. In some embodiments, immunotherapy-related toxicitycomprises CAR T-cell-related encephalopathy syndrome (CRES). In someembodiments, provided herein methods for inhibiting or reducing theincidence of a brain disease, damage or malfunction.

In some embodiments, inhibiting or reducing the incidence of CREScomprises ameliorating headaches. In some embodiments, inhibiting orreducing the incidence of CRES comprises alleviating delirium. In someembodiments, inhibiting or reducing the incidence of CRES comprisesreducing anxiety. In some embodiments, inhibiting or reducing theincidence of CRES comprises reducing tremors. In some embodiments,inhibiting or reducing the incidence of CRES comprises decreasingseizure activity. In some embodiments, inhibiting or reducing theincidence of CRES comprises decreasing confusion. In some embodiments,inhibiting or reducing the incidence of CRES comprises reducingalterations in wakefulness.

In some embodiments, inhibiting or reducing the incidence of CREScomprises reducing hallucinations. In some embodiments, inhibiting orreducing the incidence of CRES comprises reducing dysphasia. In someembodiments, inhibiting or reducing the incidence of CRES comprisesreducing ataxia. In some embodiments, inhibiting or reducing theincidence of CRES comprises reducing apraxia. In some embodiments,inhibiting or reducing the incidence of CRES comprises amelioratingfacial nerve palsy. In some embodiments, inhibiting or reducing theincidence of CRES comprises reducing motor weakness. In someembodiments, inhibiting or reducing the incidence of CRES comprisesreducing seizures. In some embodiments, inhibiting or reducing theincidence of CRES comprises reducing non-convulsive EEG seizures. Insome embodiments, inhibiting or reducing the incidence or severity ofCRES comprises improving coma recovery.

In some embodiments, inhibiting or reducing the incidence or severity ofCRES comprises reducing endothelial activation. A skilled artisan wouldappreciate that endothelial activation is an inflammatory andprocoagulant state of endothelial cells characterized by increasedinteractions with leukocytes.

In some embodiments, inhibiting or reducing the incidence of CREScomprises reducing vascular leak. The term “vascular leak” may be usedinterchangeably with the terms “vascular leak syndrome” and “capillaryleak syndrome” having all the same qualities and meanings. A skilledartisan would appreciate that vascular leak is associated withendothelial cells are separated allowing a leakage of plasma andtransendothelial migration of inflammatory cells into body tissues,resulting in tissue and organ damage. In addition, neutrophils can causemicrocirculatory occlusion, leading to decreased tissue perfusion. Insome embodiments reducing the incidence of CRES comprises reducingintravascular coagulation.

In some embodiments, inhibiting or reducing the incidence of CREScomprises reducing the concentration of at least one circulatingcytokine. In some embodiments, the cytokine is selected from a groupcomprising: hGM-CSF, IFNγ, IL-1, IL-15, IL-6, IL-8, IL-10, and IL-2. Insome embodiments, inhibiting or reducing the incidence of CRES comprisesreducing serum concentration of ANG2. In some embodiments, inhibiting orreducing the incidence of CRES comprises reducing ANG2:ANG1 ratio inserum.

In some embodiments, inhibiting or reducing the incidence of CREScomprises reducing the CRES grade. In some embodiments, inhibiting orreducing the incidence of CRES comprises improving CARTOX-10 score. Insome embodiments, inhibiting or reducing the incidence of CRES comprisesreducing a raise in intracranial pressure. In some embodiments,inhibiting or reducing the incidence of CRES comprises reducingseizures. In some embodiments, inhibiting or reducing the incidence ofCRES comprises reducing motor weakness.

In some embodiments, immunotherapy-related toxicity comprises CAR T-cellrelated CRS. In some embodiments, provided herein are methods forinhibiting or reducing the incidence or severity of CRS and/orneurotoxicity (NT).

In some embodiments, inhibiting or reducing the incidence of CRS or NTcomprises, without limitation, ameliorating fever (with or withoutrigors, malaise, fatigue, anorexia, myalgia, arthralgia, nausea,vomiting, headache, skin rash, diarrhea, tachypnea, hypoxemia, hypoxia,shock, cardiovascular tachycardia, widened pulse pressure, hypotension,capillary leak, increased early cardiac output, diminished late cardiacoutput, elevated D-dimer, hypofibrinogenemia with or without bleeding,azotemia, transaminitis, hyperbilirubinemia, mental status changes,confusion, delirium, frank aphasia, hallucinations, tremor, dysmetria,altered gait, seizures, organ failure, or any combination thereof, orany other symptom or characteristic known in the art to be associatedwith CRS.

In some embodiments, inhibiting or reducing the incidence of CRScomprises reducing the concentration of at least one circulatingcytokine. In some embodiments, the cytokine is selected from a groupcomprising: GM-CSF, IFNγ, IL-1, IL-15, IL-6, IL-8, IL-10, and IL-2.

In some embodiments, inhibiting or reducing the incidence of CRScomprises reducing the CRS grade. In some embodiments, inhibiting orreducing the incidence of NT comprises reducing the NT grade. In someembodiments, inhibiting or reducing the incidence of CRS comprisesimproving CARTOX-10 score. In some embodiments, inhibiting or reducingthe incidence of NT comprises improving CARTOX-10 score. In someembodiments, inhibiting or reducing the incidence of CRS comprisesreducing raised intracranial pressure. In some embodiments, inhibitingor reducing the incidence of CRS comprises reducing seizures. In someembodiments, inhibiting or reducing the incidence of CRS comprisesreducing motor weakness. In some embodiments, inhibiting or reducing theincidence of NT or CRS comprises inhibiting or reducing the incidence toless than 60%. In some embodiments, inhibiting or reducing the incidenceof NT or CRS comprises inhibiting or reducing the incidence to less than50%. In some embodiments, inhibiting or reducing the incidence of NT orCRS comprises inhibiting or reducing the incidence to less than 40%. Insome embodiments, inhibiting or reducing the incidence of NT or CRScomprises inhibiting or reducing the incidence to less than 30%. In someembodiments, inhibiting or reducing the incidence of NT or CRS comprisesinhibiting or reducing the incidence to less than 20% of patients. Insome embodiments, inhibiting or reducing the incidence of NT or CRScomprises eliminating NT or CRS.

In some embodiments, the subject has Grade 1 CRS and/or NT. In someembodiments, the subject has Grade 2 CRS and or NT. In some embodiments,the subject has Grade 3 CRS and/or NT. In some embodiments, the subjecthas Grade 4 CRS and/or NT. In some embodiments, the subject has anycombination of the above.

In some embodiments, inhibiting or reducing the incidence of NT or CRScomprises reducing the CRS grade, the NT grade, or both. In someembodiments, the grade is reduced to ≤3 NT and/or CRS in 95% ofpatients.

In some embodiments, the subject has a body temperature above 37° C.following immunotherapy administration. In some embodiments, the subjecthas a body temperature above 38° C. following immunotherapyadministration. In some embodiments, the subject has a body temperatureabove 39° C. following immunotherapy administration. In someembodiments, the subject has a body temperature above 40° C. followingimmunotherapy administration. In some embodiments, the subject has abody temperature above 41° C. following immunotherapy administration. Insome embodiments, the subject has a body temperature above 42° C.following immunotherapy administration.

In some embodiments, the subject has IL-6 serum concentration above 10pg/mL following immunotherapy administration. In some embodiments, thesubject has IL-6 serum concentration above 12 pg/mL followingimmunotherapy administration. In some embodiments, the subject has IL-6serum concentration above 14 pg/mL following immunotherapyadministration. In some embodiments, the subject has IL-6 serumconcentration above 16 pg/mL following immunotherapy administration. Insome embodiments, the subject has IL-6 serum concentration above 18pg/mL following immunotherapy administration. In some embodiments, thesubject has IL-6 serum concentration above 20 pg/mL followingimmunotherapy administration. In some embodiments, the subject has IL-6serum concentration above 22 pg/mL following immunotherapyadministration.

In some embodiments, the subject has an MCP-1 serum concentration above200 pg/ml following immunotherapy administration. In some embodiments,the subject has an MCP-1 serum concentration above 400 pg/ml followingimmunotherapy administration. In some embodiments, the subject has anMCP-1 serum concentration above 600 pg/ml following immunotherapyadministration. In some embodiments, the subject has an MCP-1 serumconcentration above 800 pg/ml following immunotherapy administration. Insome embodiments, the subject has an MCP-1 serum concentration above1000 pg/ml following immunotherapy administration. In some embodiments,the subject has an MCP-1 serum concentration above 1200 pg/ml followingimmunotherapy administration. In some embodiments, the subject has anMCP-1 serum concentration above 1400 pg/ml following immunotherapyadministration. In some embodiments, the subject has an MCP-1 serumconcentration above 1600 pg/ml following immunotherapy administration.In some embodiments, the subject has an MCP-1 serum concentration above1800 pg/ml following immunotherapy administration. In some embodiments,the subject has an MCP-1 serum concentration above 2000 pg/ml followingimmunotherapy administration.

In some embodiments, the subject has Grade 1 CRES. In some embodiments,the subject has Grade 2 CRES. In some embodiments, the subject has Grade3 CRES. In some embodiments, the subject has Grade 4 CRES.

In some embodiments, the subject is predisposed to have a brain disease,damage or malfunction prior to immunotherapy. In some embodiments, thepredisposition is genetic. In some embodiments, the predisposition isacquired. In some embodiments, the predisposition regards an existingmedical condition. In some embodiments, the predisposition is diagnosedprior to immunotherapy. In some embodiments, the predisposition is notdiagnosed. In some embodiments, the subject goes through medicalevaluations in order to determine predisposition to acquire animmunotherapy-related brain disease, damage or malfunction prior toimmunotherapy.

In some embodiments, medical evaluations comprise determining ANG1concentration in a body fluid. In some embodiments, medical evaluationscomprise determining ANG1 concentration in serum. In some embodiments,medical evaluations comprise determining ANG2 concentration in a bodyfluid. In some embodiments, medical evaluations comprise determiningANG2 concentration in serum. In some embodiments, medical evaluationscomprise calculating the ANG2:ANG1 ratio in serum. In some embodiments,subjects with serum ANG2:ANG1 ratio above 0.5 prior to immunotherapy arepredisposed to CRES. In some embodiments, subjects with serum ANG2:ANG1ratio above 0.7 prior to immunotherapy are predisposed to CRES. In someembodiments, subjects with serum ANG2:ANG1 ratio above 0.9 prior toimmunotherapy are predisposed to CRES. In some embodiments, subjectswith serum ANG2:ANG1 ratio above 1 prior to immunotherapy arepredisposed to CRES. In some embodiments, subjects with serum ANG2:ANG1ratio above 1.1 prior to immunotherapy are predisposed to CRES. In someembodiments, subjects with serum ANG2:ANG1 ratio above 1.3 prior toimmunotherapy are predisposed to CRES. In some embodiments, subjectswith serum ANG2:ANG1 ratio above 1.5 prior to immunotherapy arepredisposed to CRES.

In some embodiments, immunotherapy-related toxicity compriseshemophagocytic lymphohistiocytosis (HLH). In some embodiments,immunotherapy-related toxicity comprises macrophage-activation syndrome(MAS). In some embodiments, provided herein methods for inhibiting orreducing the incidence of HLH. In some embodiments, provided hereinmethods for inhibiting or reducing the incidence of MAS.

In some embodiments, inhibiting or reducing the incidence of HLHcomprises increasing survival of the subject. In some embodiments,inhibiting reducing the incidence of HLH comprises increasing time torelapse. In some embodiments, inhibiting or reducing the incidence ofMAS comprises increasing survival of the subject. In some embodiments,inhibiting reducing the incidence of MAS comprises increasing time torelapse.

In some embodiments, inhibiting or reducing the incidence of HLH or MAScomprises inhibiting macrophage activation and/or proliferation. In someembodiments, inhibiting or reducing the incidence of HLH or MAScomprises inhibiting T lymphocytes activation and/or proliferation. Insome embodiments, inhibiting or reducing the incidence of HLH or MAScomprises reducing the concentration of circulating IFNγ. In someembodiments, inhibiting or reducing the incidence of HLH or MAScomprises reducing the concentration of circulating of GM-CSF.

In some embodiments the subject presents with fever followingimmunotherapy. In some embodiments the subject presents withsplenomegaly following immunotherapy. In some embodiments the subjectpresents with cytopenia following immunotherapy. In some embodiments thesubject presents with cytopenia in two or more cell lines followingimmunotherapy. In some embodiments the subject presents withhypertriglyceridemia following immunotherapy. In some embodiments thesubject presents with hypofibrinogenemia following immunotherapy. Insome embodiments the subject presents with hemophagocytosis followingimmunotherapy. In some embodiments hemophagocytosis is observed in bonemarrow. In some embodiments the subject presents with low NK-cellactivity following immunotherapy. In some embodiments the subjectpresents with absent NK activity following immunotherapy.

In some embodiments the subject presents with ferritin serumconcentrations above 100 U/ml following immunotherapy. In someembodiments the subject presents with ferritin serum concentrationsabove 300 U/ml following immunotherapy. In some embodiments the subjectpresents with ferritin serum concentrations above 500 U/ml followingimmunotherapy. In some embodiments the subject presents with ferritinserum concentrations above 700 U/ml following immunotherapy. In someembodiments the subject presents with ferritin serum concentrationsabove 900 U/ml following immunotherapy.

In some embodiments the subject presents with soluble CD25 serumconcentration above 1200 U/ml following immunotherapy. In someembodiments the subject presents with soluble CD25 serum concentrationabove 1500 U/ml following immunotherapy. In some embodiments the subjectpresents with soluble CD25 serum concentration above 1800 U/ml followingimmunotherapy. In some embodiments the subject presents with solubleCD25 serum concentration above 2000 U/ml following immunotherapy. Insome embodiments the subject presents with soluble CD25 serumconcentration above 2200 U/ml following immunotherapy. In someembodiments the subject presents with soluble CD25 serum concentrationabove 2400 U/ml following immunotherapy. In some embodiments the subjectpresents with soluble CD25 serum concentration above 2700 U/ml followingimmunotherapy. In some embodiments the subject presents with solubleCD25 serum concentration above 3000 U/ml following immunotherapy.

In some embodiments, the subject is predisposed to have HLH. In someembodiments, the predisposition is genetic. In some embodiments, thepredisposition regards an existing medical condition. A skilled artisanwould appreciate that sporadic HLH has been associated with a number ofgenetic mutations. In some embodiments, the subject carries a mutationin a gene selected from PRF1, UNC13D, STX11, STXBP2, or RAB27A, or anycombination thereof. In some embodiments, the subject has reduced orabsent expression of perforin.

hGM-CSF Antagonists

hGM-CSF antagonists suitable for use selectively interfere with theinduction of signaling by the hGM-CSF receptor by causing a reduction inthe binding of hGM-CSF to the receptor. Such antagonists may includeantibodies that bind the hGM-CSF receptor, antibodies that bind tohGM-CSF, GM-CSF analogs such as E21R, and other proteins or smallmolecules that compete for binding of hGM-CSF to its receptor or inhibitsignaling that normally results from the binding of the ligand to thereceptor.

In many embodiments, the hGM-CSF antagonist used in the invention is apolypeptide e.g., an anti-hGM-CSF antibody, an anti-hGM-CSF receptorantibody, a soluble hGM-CSF receptor, or a modified GM-CSF polypeptidethat competes for binding with hGM-CSF to a receptor, but is inactive.Such proteins are often produced using recombinant expressiontechnology. Such methods are widely known in the art. General molecularbiology methods, including expression methods, can be found, e.g., ininstruction manuals, such as, Sambrook and Russell (2001) MolecularCloning: A laboratory manual 3rd ed. Cold Spring Harbor LaboratoryPress; Current Protocols in Molecular Biology (2006) John Wiley and SonsISBN: 0-471-50338-X.

A variety of prokaryotic and/or eukaryotic based protein expressionsystems may be employed to produce a hGM-CSF antagonist protein. Manysuch systems are widely available from commercial suppliers.

hGM-CSF Antibodies

The hGM-CSF antibodies of the present invention are antibodies that bindwith high affinity to hGM-CSF and are antagonists of hGM-CSF. Theantibodies comprise variable regions with a high degree of identity tohuman germ-line V_(H) and V_(L) sequences.

In preferred embodiments, the BSD sequence in CDRH3 of an antibody ofthe invention comprises the amino acid sequence RQRFPY or RDRFPY. TheBSD in CDRL3 comprises FNK or FNR.

Complete V-regions are generated in which the BSD forms part of the CDR3and additional sequences are used to complete the CDR3 and add a FR4sequence. Typically, the portion of the CDR3 excluding the BSD and thecomplete FR4 are comprised of human germ-line sequences. In someembodiments, the CDR3-FR4 sequence excluding the BSD differs from humangerm-line sequences by not more than 2 amino acids on each chain. Insome embodiments, the J-segment comprises a human germline J-segment.Human germline sequences can be determined, for example, through thepublicly available international ImMunoGeneTics database (IMGT) andV-base (on the worldwide web at vbase.mrc-cpe.cam.ac.uk).

The human germline V-segment repertoire consists of 51 heavy chainV-regions, 40 K light chain V-segments, and 31 λ light chain V-segments,making a total of 3,621 germline V-region pairs, in addition, there arestable allelic variants for most of these V-segments, but thecontribution of these variants to the structural diversity of thegermline repertoire is limited. The sequences of all human germ-lineV-segment genes are known and can be accessed in the V-base database,provided by the MRC Centre for Protein Engineering, Cambridge, UnitedKingdom (see, also Chothia et al., 1992, J Mol Biol 227:776-798;Tomlinson et al., 1995, EMBO J 14:4628-4638; and Williams et al., 1996,J Mol Biol 264:220-232).

Antibodies or antibodies fragments as described herein can be expressedin prokaryotic or eukaryotic microbial systems or in the cells of highereukaryotes such as mammalian cells.

An antibody that is employed in the invention can be in any format. Forexample, in some embodiments, the antibody can be a complete antibodyincluding a constant region, e.g., a human constant region, or can be afragment or derivative of a complete antibody, e.g., an Fd, a Fab, Fab′,F(ab′)₂, scFv, Fv, an Fv fragment, or a single domain antibody, such asa nanobody or a camelid antibody. Such antibodies may additionally berecombinantly engineered by methods well known to persons of skill inthe art. As noted above, such antibodies can be produced using knowntechniques.

In some embodiments, the hGM-CSF antagonist is an antibody that binds tohGM-CSF or an antibody that binds to the hGM-CSF receptor α or βsubunit. The antibodies can be raised against hGM-CSF (or hGM-CSFreceptor) proteins, or fragments, or produced recombinantly. Antibodiesto GM-CSF for use in the invention can be neutralizing or can benon-neutralizing antibodies that bind GM-CSF and increase the rate of invivo clearance of hGM-CSF such that the hGM-CSF level in the circulationis reduced. Often, the hGM-CSF antibody is a neutralizing antibody.

Methods of preparing polyclonal antibodies are known to the skilledartisan (e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988);Methods in Immunology). Polyclonal antibodies can be raised in a mammalby one or more injections of an immunizing agent and, if desired, anadjuvant. The immunizing agent includes a GM-CSF or GM-CSF receptorprotein, e.g., a human GM-CSF or GM-CSF receptor protein, or fragmentthereof.

In some embodiment, a GM-CSF antibody for use in the invention ispurified from human plasma. In such embodiments, the GM-CSF antibody istypically a polyclonal antibody that is isolated from other antibodiespresent in human plasma. Such an isolation procedure can be performed,e.g., using known techniques, such as affinity chromatography.

In some embodiments, the GM-CSF antagonist is a monoclonal antibody.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler & Milstein, Nature 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent, such as human GM-CSF, toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro. The immunizing agent preferablyincludes human GM-CSF protein, fragments thereof, or fusion proteinthereof.

Human monoclonal antibodies can be produced using various techniquesknown in the art, including phage display libraries (Hoogenboom &Winter, J. MoI. Biol. 227:381 (1991); Marks et al, J. MoI. Biol. 222:581(1991)). The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, p. 77 (1985) and Boerneret al., J. Immunol. 147(1):86-95 (1991)). Similarly, human antibodiescan be made by introducing of human immunoglobulin loci into transgenicanimals, e.g., mice in which the endogenous immunoglobulin genes havebeen partially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire. This approach is described, e.g., in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inthe following scientific publications: Marks et al., Bio/Technology10:779-783 (1992); Lonberg et al, Nature 368:856-859 (1994); Morrison,Nature 368:812-13 (1994); Fishwild et al, Nature Biotechnology 14:845-51(1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg & Huszar,Intern. Rev. Immunol. 13:65-93 (1995).

In some embodiments the anti-GM-CSF antibodies are chimeric or humanizedmonoclonal antibodies. As noted supra, humanized forms of antibodies arechimeric immunoglobulins in which residues from a complementarydetermining region (CDR) of human antibody are replaced by residues froma CDR of a non-human species such as mouse, rat or rabbit having thedesired specificity, affinity and capacity.

In some embodiments of the invention, the antibody is additionallyengineered to reduced immunogenicity, e.g., so that the antibody issuitable for repeat administration. Methods for generating antibodieswith reduced immunogenicity include humanization/humaneering proceduresand modification techniques such as de-immunization, in which anantibody is further engineered, e.g., in one or more framework regions,to remove T cell epitopes.

In some embodiments, the antibody is a humaneered antibody. A humaneeredantibody is an engineered human antibody having a binding specificity ofa reference antibody, obtained by joining a DNA sequence encoding abinding specificity determinant (BSD) from the CDR3 region of the heavychain of the reference antibody to human VH segment sequence and a lightchain CDR3 BSD from the reference antibody to a human VL segmentsequence. Methods for Humaneering are provided in US patent applicationpublication no. 20050255552 and US patent application publication no.20060134098. Methods for signal-less secretion of antibody fragmentsfrom E. coli are described in US patent application 20070020685.

An antibody can further be de-immunized to remove one or more predictedT-cell epitopes from the V-region of an antibody. Such procedures aredescribed, for example, in WO 00/34317.

The heavy chain constant region is often a gamma chain constant region,for example, a gamma-1, gamma-2, gamma-3, or gamma-4 constant region. Insome embodiments, e.g., where the antibody is a fragment, the antibodycan be conjugated to another molecule, e.g., to provide an extendedhalf-life in vivo such as a polyethylene glycol (pegylation) or serumalbumin. Examples of PEGylation of antibody fragments are provided inKnight et al (2004) Platelets 15: 409 (for abciximab); Pedley et al(1994) Br. J. Cancer 70: 1126 (for an anti-CEA antibody) Chapman et al(1999) Nature Biotech. 17: 780.

An antibody for use in the invention binds to hGM-CSF or hGM-CSFreceptor. Any number of techniques can be used to determine antibodybinding specificity. See, e.g., Harlow & Lane, Antibodies, A LaboratoryManual (1988) for a description of immunoassay formats and conditionsthat can be used to determine specific immunoreactivity of an antibody.

An exemplary antibody suitable for use with the present invention isc19/2 (a human mouse chimeric anti-hGM-CSF antibody). In someembodiments, a monoclonal antibody that competes for binding to the sameepitope as c19/2, or that binds the same epitope as c19/2, is used. Theability of a particular antibody to recognize the same epitope asanother antibody is typically determined by the ability of the firstantibody to competitively inhibit binding of the second antibody to theantigen. Any of a number of competitive binding assays can be used tomeasure competition between two antibodies to the same antigen. Forexample, a sandwich ELISA assay can be used for this purpose. This iscarried out by using a capture antibody to coat the surface of a well. Asubsaturating concentration of tagged-antigen is then added to thecapture surface. This protein will be bound to the antibody through aspecific antibody:epitope interaction. After washing a second antibody,which has been covalently linked to a detectable moiety (e.g., HRP, withthe labeled antibody being defined as the detection antibody) is addedto the ELISA. If this antibody recognizes the same epitope as thecapture antibody it will be unable to bind to the target protein as thatparticular epitope will no longer be available for binding. If howeverthis second antibody recognizes a different epitope on the targetprotein it will be able to bind and this binding can be detected byquantifying the level of activity (and hence antibody bound) using arelevant substrate. The background is defined by using a single antibodyas both capture and detection antibody, whereas the maximal signal canbe established by capturing with an antigen specific antibody anddetecting with an antibody to the tag on the antigen. By using thebackground and maximal signals as references, antibodies can be assessedin a pair-wise manner to determine epitope specificity.

A first antibody is considered to competitively inhibit binding of asecond antibody, if binding of the second antibody to the antigen isreduced by at least 30%, usually at least about 40%, 50%, 60% or 75%,and often by at least about 90%, in the presence of the first antibodyusing any of the assays described above.

In some embodiments of the invention, an antibody is employed thatcompetes with binding, or bind, to the same epitope as a known antibody,e.g., c19/2. Method of mapping epitopes are well known in the art. Forexample, one approach to the localization of functionally active regionsof human granulocyte-macrophage colony-stimulating factor (hGM-CSF) isto map the epitopes recognized by neutralizing anti-hGM-CSF monoclonalantibodies. For example, the epitope to which c19/2 (which has the samevariable regions as the neutralizing antibody LMM 102) binds has beendefined using proteolytic fragments obtained by enzymic digestion ofbacterially synthesized hGM-CSF (Dempsey, et al, Hybridoma 9:545-558,1990). RP-HPLC fractionation of a tryptic digest resulted in theidentification of an immunoreactive “tryptic core” peptide containing 66amino acids (52% of the protein). Further digestion of this “trypticcore” with S. aureus V8 protease produced a unique immunoreactivehGM-CSF product comprising two peptides, residues 86-93 and 112-127,linked by a disulfide bond between residues 88 and 121. The individualpeptides were not recognized by the antibody.

In some embodiments, the antibodies suitable for use with the presentinvention have a high affinity binding for human GM-CSF or hGM-CSFreceptor. High affinity binding between an antibody and an antigenexists if the dissociation constant (KD) of the antibody is <about 10nM, typically <1 nM, and preferably <100 pM. In some embodiments, theantibody has a dissociation rate of about 10⁻⁴ per second or better.

A variety of methods can be used to determine the binding affinity of anantibody for its target antigen such as surface plasmon resonanceassays, saturation assays, or immunoassays such as ELISA or RIA, as arewell known to persons of skill in the art. An exemplary method fordetermining binding affinity is by surface plasmon resonance analysis ona BIAcore™ 2000 instrument (Biacore AB, Freiburg, Germany) using CMSsensor chips, as described by Krinner et al, (2007) MoI. Immunol.February; 44(5):916-25. (Epub 2006 May H)).

In some embodiments, the hGM-CSF antagonists are neutralizing antibodiesto hGM-CSF, its receptor or its receptor subunit, which bind in a mannerthat interferes with the binding of hGM-CSF to its receptor or receptorsubunit. In some embodiments, an anti-hGM-CSF antibody for use in theinvention inhibits binding to the alpha subunit of the hGM-CSF receptor.Such an antibody can, for example, bind to hGM-CSF at the region wherehGM-CSF binds to the receptor and thereby inhibit binding. In anotherembodiments, the anti-hGM-CSF antibody inhibits hGM-CSF functioningwithout blocking its binding to the alpha subunit of the hGM-CSFreceptor.

II. Heavy Chains

A heavy chain of an anti-hGM-CSF antibody of the invention comprises aheavy-chain V-region that comprises the following elements:

1) human heavy-chain V-segment sequences comprisingFR1-CDR1-FR2-CDR2-FR3

2) a CDRH3 region comprising the amino acid sequence R(Q/D)RFPY

3) a FR4 contributed by a human germ-line J-gene segment.

Examples of V-segment sequences that support binding to hGM-CSF incombination with a CDR3-FR4 segment described above together with acomplementary V_(L) region are shown in FIG. 1. The V-segments can be,e.g., from the human VH1 subclass. In some embodiments, the V-segment isa human V_(H)1 sub-class segment that has a high degree of amino-acidsequence identity, e.g., at least 80%, 85%, or 90% or greater identity,to the germ-line segment VH1 1-02 or VH1 1-03. In some embodiments, theV-segment differs by not more than 15 residues from VH1 1-02 or VH1 1-03and preferably not more than 7 residues.

The FR4 sequence of the antibodies of the invention is provided by ahuman JH1, JH3, JH4, JH5 or JH6 gene germline segment, or a sequencethat has a high degree of amino-acid sequence identity to a humangermline JH segment. In some embodiments, the J segment is a humangermline JH4 sequence.

The CDRH3 also comprises sequences that are derived from a humanJ-segment. Typically, the CDRH3-FR4 sequence excluding the BSD differsby not more than 2 amino acids from a human germ-line J-segment. Intypical embodiments, the J-segment sequences in CDRH3 are from the sameJ-segment used for the FR4 sequences. Thus, in some embodiments, theCDRH3-FR4 region comprises the BSD and a complete human JH4 germ-linegene segment. An exemplary combination of CDRH3 and FR4 sequences isshown below, in which the BSD is in bold and human germ-line J-segmentJH4 residues are underlined:

CDR3 . R(Q/D)RFPY YFDYWGQGTLVTVSS

In some embodiments, an antibody of the invention comprises a V-segmentthat has at least 90% identity, or at least 91%, 92% 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identity to the germ-line segment VH 1-02 orVH1-03; or to one of the V-segments of the V_(H) regions shown in FIG.1, such as a V-segment portion of VH#1, VH#2, VH#3, VH#4, or VH#5.

In some embodiments, the V-segment of the V_(H) region has a CDR1 and/orCDR2 as shown in FIG. 1. For example, an antibody of the invention mayhave a CDR1 that has the sequence GYYMH or NYYIH; or a CDR2 that has thesequence WINPNSGGTNYAQKFQG or WINAGNGNTKYSQKFQG.

In particular embodiments, an antibody has both a CDR1 and a CDR2 fromone of the V_(H) region V-segments shown in FIG. 1 and a CDR3 thatcomprises R(Q/D)RFPY, e.g., RDRFPYYFDY or RQRFPYYFDY. Thus, in someembodiments, an anti-GM-CSF antibody of the invention, may for example,have a CDR3-FR4 that has the sequence R(Q/D)RFPYYFDYWGQGTLVTVSS and aCDR1 and/or CDR2 as shown in FIG. 1.

In some embodiments, a V_(H) region of an antibody of the invention hasa CDR3 that has a binding specificity determinant R(Q/D)RFPY, a CDR2from a human germline VH1 segment or a CDR1 from a human germline VH1.In some embodiments, both the CDR1 and CDR2 are from human germline VH1segments.

III. Light Chains

A light chain of an anti-hGM-CSF antibody of the invention comprises atlight-chain V-region that comprises the following elements:

1) human light-chain V-segment sequences comprisingFR1-CDR1-FR2-CDR2-FR3

2) a CDRL3 region comprising the sequence FNK or FNR, e.g., QQFNRSPLT orQQFNKSPLT.

3) a FR4 contributed by a human germ-line J-gene segment.

The V_(L) region comprises either a Vlambda or a Vkappa V-segment. Anexample of a Vkappa sequence that supports binding in combination with acomplementary V_(H)-region is provided in FIG. 1.

The V_(L) region CDR3 sequence comprises a J-segment derived sequence.In typical embodiments, the J-segment sequences in CDRL3 are from thesame J-segment used for FR4. Thus, the sequence in some embodiments maydiffer by not more than 2 amino acids from human kappa germ-lineV-segment and J-segment sequences. In some embodiments, the CDRL3-FR4region comprises the BSD and the complete human JK4 germline genesegment. Exemplary CDRL3-FR4 combinations for kappa chains are shownbelow in which the minimal essential binding specificity determinant isshown in bold and JK4 sequences are underlined:

CDR3 QQFNRSPLTFGGGTKVEIK QQFNKSPLTFGGGTKVEIK

The Vkappa segments are typically of the VKIII sub-class. In someembodiments, the segments have at least 80% sequence identity to a humangermline VKIII subclass, e.g., at least 80% identity to the humangerm-line VKIIIA27 sequence. In some embodiments, the Vkappa segment maydiffer by not more than 18 residues from VKIIIA27. In other embodiments,the V_(L) region V-segment of an antibody of the invention has at least85% identity, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identity to the human kappa V-segment sequence of a V_(L)region shown in FIG. 1, for example, the V-segment sequence of VK#1,VK#2, VK#3, or VK#4.

In some embodiments, the variable region is comprised of human V-genesequences. For example, a variable region sequence can have at least 80%identity, or at least 85% identity, at least 90% identity, at least 95%identity, at least 96% identity, at least 97% identity, at least 98%identity, or at least 99% identity, or greater, with a human germ-lineV-gene sequence.

In some embodiments, the V-segment of the V_(L) region has a CDR1 and/orCDR2 as shown in FIG. 1. For example, an antibody of the invention mayhave a CDR1 sequence of RASQSVGTNVA or RASQSIGSNLA; or a CDR2 sequenceSTSSRAT.

In particular embodiments, an anti-GM-CSF antibody of the invention mayhave a CDR1 and a CDR2 in a combination as shown in one of theV-segments of the V_(L) regions set forth in FIG. 1 and a CDR3 sequencethat comprises FNK or FNR, e.g., the CDR3 may be QQFNKSPLT or QQFNRSPLT.In some embodiments, such a GM-CSF antibody may comprise an FR4 regionthat is FGGGTKVEIK. Thus, an anti-GM-CSF antibody of the invention, cancomprise, e.g., both the CDR1 and CDR2 from one of the V_(L) regionsshown in FIG. 1 and a CDR3-FR4 region that is FGGGTKVEIK.

IV. Preparation of hGM-CSF Antibodies

An antibody of the invention may comprise any of the V_(H) regions VH#1,VH#2, VH#3, VH#4, or VH#5 as shown in FIG. 1. In some embodiment, anantibody of the invention may comprise any of the V_(L) regions VK#1,VK#2, VK#3, or VK#4 as shown in FIG. 1. In some embodiments, theantibody has a V_(H) region VH#1, VH#2, VH#3, VH#4, or VH#5 as shown inFIG. 1; and a V_(L) region VK#1, VK#2, VK#3, or VK#4 as shown in FIG. 1,as described, e.g., in U.S. Pat. Nos. 8,168,183 and 9,017,674, each ofwhich is incorporated herein by reference in its entirety.

An antibody may be tested to confirm that the antibody retains theactivity of antagonizing hGM-CSF activity. The antagonist activity canbe determined using any number of endpoints, including proliferationassays. Neutralizing antibodies and other hGM-CSF antagonists may beidentified or evaluated using any number of assays that assess hGM-CSFfunction. For example, cell-based assays for hGM-CSF receptor signaling,such as assays which determine the rate of proliferation of ahGM-CSF-dependent cell line in response to a limiting amount of hGM-CSF,are conveniently used. The human TF-1 cell line is suitable for use insuch an assay. See, Krinner et al., (2007) Mol. Immunol. In someembodiments, the neutralizing antibodies of the invention inhibithGM-CSF stimulated TF-I cell proliferation by at least 50%, when ahGM-CSF concentration is used which stimulates 90% maximal TF-I cellproliferation. Thus, typically, a neutralizing antibody, or otherhGM-CSF antagonist for use in the invention, has an EC50 of less than 10nM (e.g., Table 2). Additional assays suitable for use in identifyingneutralizing antibodies suitable for use with the present invention willbe well known to persons of skill in the art. In other embodiments, theneutralizing antibodies inhibit hGM-CSF stimulated proliferation by atleast about 75%, 80%, 90%, 95%, or 100%, of the antagonist activity ofthe antibody chimeric c19/2, e.g., WO03/068920, which has the variableregions of the mouse monoclonal antibody LMM102 and the CDRs.

An exemplary chimeric antibody suitable for use as a hGM-CSF antagonistis c19/2. The c 19/2 antibody binds hGM-CSF with a monovalent bindingaffinity of about 10 pM as determined by surface plasmon resonanceanalysis. The heavy and light chain variable region sequences of c19/2are known (e.g., WO03/068920). The CDRs, as defined according to Kabat,are:

CDRH1 DYNIH CDRH2 YIAPYSGGTGYNQEFKN CDRH3 RDRFPYYFDY CDRL1 KASQNVGSNVACDRL2 SASYRSG CDRL3 QQFNRSPLT.

The CDRs can also be determined using other well-known definitions inthe art, e.g., Chothia, international ImMunoGeneTics database (IMGT),and AbM.

In some embodiments, an antibody used in the invention competes forbinding to, or binds to, the same epitope as c19/2. The GM-CSF epitoperecognized by c19/2 has been identified as a product that has twopeptides, residues 86-93 and residues 112-127, linked by a disulfidebond between residues 88 and 121. The c19/2 antibody inhibits theGM-CSF-dependent proliferation of a human TF-I leukemia cell line withan EC50 of 30 pM when the cells are stimulated with 0.5 ng/ml GM-CSF. Insome embodiments, the antibody used in the invention binds to the sameepitope as c19/2.

An antibody for administration, such as c19/2, can be additionallyHumaneered. For example, the c19/2 antibody can be further engineered tocontain human V gene segments.

A high-affinity antibody may be identified using well known assays todetermine binding activity and affinity. Such techniques include ELISAassays as well as binding determinations that employ surface plasmonresonance or interferometry. For example, affinities can be determinedby biolayer interferometry using a ForteBio (Mountain View, Calif.)Octet biosensor. An antibody of the invention typically binds withsimilar affinity to both glycosylated and non-glycosylated form ofhGM-CSF.

Antibodies of the invention compete with c19/2 for binding to hGM-CSF.The ability of an antibody described herein to block or compete withc19/2 for binding to hGM-CSF indicates that the antibody binds to thesame epitope c19/2 or to an epitope that is close to, e.g., overlapping,with the epitope that is bound by c19/2. In other embodiments anantibody described herein, e.g., an antibody comprising a V_(H) andV_(L) region combination as shown in the table provided in FIG. 1, canbe used as a reference antibody for assessing whether another antibodycompetes for binding to hGM-CSF. A test antibody is considered tocompetitively inhibit binding of a reference antibody, if binding of thereference antibody to the antigen is reduced by at least 30%, usually atleast about 40%, 50%, 60% or 75%, and often by at least about 90%, inthe presence of the test antibody. Many assays can be employed to assessbinding, including ELISA, as well as other assays, such as immunoblots.In some embodiments, an antibody of the invention has a dissociationrate that is at least 2 to 3-fold slower than a reference chimeric c19/2monoclonal antibody assayed under the same conditions, but has a potencythat is at least 6-10 times greater than that of the reference antibodyin neutralizing hGM-CSF activity in a cell-based assay that measureshGM-CSF activity.

Methods for the isolation of antibodies with V-region sequences close tohuman germ-line sequences have previously been described (US patentapplication publication nos. 20050255552 and 20060134098). Antibodylibraries may be expressed in a suitable host cell including mammaliancells, yeast cells or prokaryotic cells. For expression in some cellsystems, a signal peptide can be introduced at the N-terminus to directsecretion to the extracellular medium. Antibodies may be secreted frombacterial cells such as E. coli with or without a signal peptide.Methods for signal-less secretion of antibody fragments from E. coli aredescribed in US patent application 20070020685.

In some embodiments, an hGM-CSF-binding antibody of the invention isgenerated where, an antibody that has a CDR from one of the VH-regionsof the invention shown in FIG. 1, is combined with an antibody having aCDR of one of the V_(L)-regions shown in FIG. 1, and expressed in any ofa number of formats in a suitable expression system. Thus, the antibodymay be expressed as a scFv, Fab, Fab′ (containing an immunoglobulinhinge sequence), F(ab′)₂, (formed by di-sulfide bond formation betweenthe hinge sequences of two Fab′ molecules), whole immunoglobulin ortruncated immunoglobulin or as a fusion protein in a prokaryotic oreukaryotic host cell, either inside the host cell or by secretion. Amethionine residue may optionally be present at the N-terminus, forexample, in polypeptides produced in signal-less expression systems.Each of the V_(H)-regions described herein may be paired with each ofthe V_(L) regions to generate an anti-hGM-CSF antibody. In anembodiment, a fusion protein comprises an anti-hGM-CSF-binding antibodyof the invention or a fragment thereof (in non-limiting examples, ananti-hGM-CSF antibody fragment is a Fab, a Fab′, a F(ab′)2, a scFv, or adAB), and human transferrin, wherein the human transferrin is fused tothe antibody at the end of the heavy chain constant region 1 (C_(H)1),after the hinge, or after C_(H)3, as described in Shin, S-U., et al.Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 2820-2824, 1995, which isincorporated herein by reference in its entirety.

Exemplary combinations of heavy and light chains are shown in the tableprovided in FIG. 1. In some embodiment, the antibody VL region, e.g.,VK#1, VK#2, VK#3, or VK#4 of FIG. 1, is combined with a human kappaconstant region to form the complete light-chain. Further, in someembodiments, the VH region is combined a human gamma-1 constant regions.Any suitable gamma-1 allotype can be chose, such as the f-allotype.Thus, in some embodiments, the antibody is an IgG, e.g., having anf-allotype, that has a VH selected from VH#1, VH#2, VH#3, VH#4, or VH#5(FIG. 1), and a VL selected from VK#1, VK#2, VK#3, or VK#4 (FIG. 1).

The antibodies of the invention inhibit hGM-CSF receptor activation,e.g., by inhibiting hGM-CSF binding to the receptor, and exhibit highaffinity binding to hGM-CSF, e.g., 500 pM. In some embodiments, theantibody has a dissociation constant of about 10⁻⁴ per sec or less. Notto be bound by theory, an antibody with a slower dissociation constantprovides improved therapeutic benefit. For example, an antibody of theinvention that has a three-fold slower off-rate than c19/2, produced a10-fold more potent hGM-CSF neutralizing activity, e.g., in a cell-basedassay such as IL-8 production (see, e.g., Example 2).

Antibodies may be produced using any number of expression systems,including both prokaryotic and eukaryotic expression systems. In someembodiments, the expression system is a mammalian cell expression, suchas a CHO cell expression system. Many such systems are widely availablefrom commercial suppliers. In embodiments in which an antibody comprisesboth a V_(H) and V_(L) region, the V_(H) and V_(L) regions may beexpressed using a single vector, e.g., in a dicistronic expression unit,or under the control of different promoters. In other embodiments, theV_(H) and V_(L) region may be expressed using separate vectors. A V_(H)or V_(L) region as described herein may optionally comprise a methionineat the N-terminus.

An antibody of the invention may be produced in any number of formats,including as a Fab, a Fab′, a F(ab′)₂, a scFv, or a dAB. An antibody ofthe invention can also include a human constant region. The constantregion of the light chain may be a human kappa or lambda constantregion. The heavy chain constant region is often a gamma chain constantregion, for example, a gamma-1, gamma-2, gamma-3, or gamma-4 constantregion. In other embodiments, the antibody may be an IgA.

In some embodiments of the invention, the antibody V_(L) region, e.g.,VK#1, VK#2, VK#3, or VK#4 of FIG. 1, is combined with a human kappaconstant region (e.g., SEQ ID NO:10) to form the complete light-chain.

In some embodiments of the invention, the VH region is combined a humangamma-1 constant region. Any suitable gamma-1 f allotype can be chosen,such as the f-allotype. Thus, in some embodiments, the antibody is anIgG having an f-allotype constant region, e.g., SEQ ID NO:11, that has aVH selected from VH#1, VH#2, VH#3, VH#4, or

VH#5 (FIG. 1). In some embodiments, the antibody has a V_(L) selectedfrom VK#1, VK#2, VK#3, or VK#4 (FIG. 1.) In particular embodiments, theantibody has a kappa constant region as set forth in SEQ ID NO:10, and aheavy chain constant region as set forth in SEQ ID NO:11, where theheavy and light chain variable regions comprise one of the followingcombinations from the sequences set forth in FIG. 1: a) VH#2, VK#3; b)VH#1, VK#3; c) VH#3, VK#1; d) VH#3, VL#3; e) VH#4, VK#4; f) VH#4, VK#2;g) VH#5, VK#1; h) VH#5, VK#2; i) VH#3, VK#4; or j) VH#3, VL#3).

In some embodiments, e.g., where the antibody is a fragment, theantibody can be conjugated to another molecule, e.g., polyethyleneglycol (PEGylation) or serum albumin, to provide an extended half-lifein vivo. Examples of PEGylation of antibody fragments are provided inKnight et al. Platelets 15:409, 2004 (for abciximab); Pedley et al., Br.J. Cancer 70:1126, 1994 (for an anti-CEA antibody); Chapman et al.,Nature Biotech. 17:780, 1999; and Humphreys, et al., Protein Eng. Des.20: 227, 2007).

In some embodiments, the antibodies of the invention are in the form ofa Fab′ fragment. A full-length light chain is generated by fusion of aV_(L)-region to human kappa or lambda constant region. Either constantregion may be used for any light chain; however, in typical embodiments,a kappa constant region is used in combination with a Vkappa variableregion and a lambda constant region is used with a Vlambda variableregion.

The heavy chain of the Fab′ is a Fd′ fragment generated by fusion of aV_(H)-region of the invention to human heavy chain constant regionsequences, the first constant (CH1) domain and hinge region. The heavychain constant region sequences can be from any of the immunoglobulinclasses, but is often from an IgG, and may be from an IgG1, IgG2, IgG3or IgG4. The Fab′ antibodies of the invention may also be hybridsequences, e.g., a hinge sequence may be from one immunoglobulinsub-class and the CH1 domain may be from a different sub-class.

V. Administration of Anti-hGM-CSF Antibodies for the Treatment ofDiseases in which GM-CSF is a Target.

The invention also provides methods of treating a patient that has adisease involving hGM-CSF in which it is desirable to inhibit hGM-CSFactivity, i.e., in which hGM-CSF is a therapeutic target. In someembodiments, such a patient has a chronic inflammatory disease, e.g.,arthritis, e.g., rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis, juvenile idiopathic arthritis, systemic-onset Still'sdisease and other inflammatory diseases of the joints; inflammatorybowel diseases, e.g., ulcerative colitis, Crohn's disease, Barrett'ssyndrome, ileitis, enteritis, eosinophilic esophagitis andgluten-sensitive enteropathy; inflammatory disorders of the respiratorysystem, such as asthma, eosinophilic asthma, adult respiratory distresssyndrome, allergic rhinitis, silicosis, chronic obstructive pulmonarydisease, hypersensitivity lung diseases, interstitial lung disease,diffuse parenchymal lung disease, bronchiectasis; inflammatory diseasesof the skin, including psoriasis, scleroderma, and inflammatorydermatoses such as eczema, atopic dermatitis, urticaria, and pruritis;disorders involving inflammation of the central and peripheral nervoussystem, including multiple sclerosis, idiopathic demyelinatingpolyneuropathy, Guillain-Barre syndrome, chronic inflammatorydemyelinating polyneuropathy, neurofibromatosis and neurodegenerativediseases such as Alzheimer's disease. Various other inflammatorydiseases can be treated using the methods of the invention. Theseinclude systemic lupus erythematosis, immune-mediated renal disease,e.g., glomerulonephritis, and spondyloarthropathies; and diseases withan undesirable chronic inflammatory component such as systemicsclerosis, idiopathic inflammatory myopathies, Sjogren's syndrome,vasculitis, sarcoidosis, thyroiditis, gout, otitis, conjunctivitis,sinusitis, sarcoidosis, Behcet's syndrome, autoimmunelymphoproliferative syndrome (or ALPS, also known as Canale-Smithsyndrome), Ras-associated autoimmune leukoproliferative disorder (orRALD), Noonan syndrome, hepatobiliary diseases such as hepatitis,primary biliary cirrhosis, granulomatous hepatitis, and sclerosingcholangitis. In some embodiments, the patient has inflammation followinginjury to the cardiovascular system. Various other inflammatory diseasesinclude Kawasaki's disease, Multicentric Castleman's Disease,tuberculosis and chronic cholecystitis. Additional chronic inflammatorydiseases are described, e.g., in Harrison's Principles of InternalMedicine, 12th Edition, Wilson, et al., eds., McGraw-Hill, Inc.). Insome embodiments, a patient treated with an antibody has a cancer inwhich GM-CSF contributes to tumor or cancer cell growth, including butnot limited to, e.g., acute myeloid leukemia, plexiformneurofibromatosis, autoimmune lymphoproliferative syndrome (or ALPS,also known as Canale-Smith syndrome), Ras-associated autoimmuneleukoproliferative disorder (or RALD), Noonan syndrome, chronicmyelomonocytic leukemia, juvenile myelomonocytic leukemia, and acutemyeloid leukemia. In some embodiments, a patient treated with anantibody of the invention has, or is at risk of heart failure, e.g., dueto ischemic injury to the cardiovascular system such as ischemic heartdisease, stroke, and atherosclerosis. In some embodiments, a patienttreated with an antibody of the invention has asthma. In someembodiments, a patient treated with an antibody of the invention hasAlzheimer's disease. In some embodiments, a patient treated with anantibody of the invention has osteopenia, e.g., osteoporosis. In someembodiments, a patient treated with an antibody of the invention hasthrombocytopenia purpura. In some embodiments, the patient has Type I orType II diabetes. In some embodiments, a patient may have more than onedisease in which GM-CSF is a therapeutic target, e.g., a patient mayhave rheumatoid arthritis and heart failure, or osteoporosis andrheumatoid arthritis, etc.

Two other examples of neutralizing anti-GM-CSF antibody are the humanElO antibody and human G9 antibody described in Li et al, (2006) PNAS103(10):3557-3562. ElO and G9 are IgG class antibodies. ElO has an 870pM binding affinity for GM-CSF and G9 has a 14 pM affinity for GM-CSF.Both antibodies are specific for binding to human GM-CSF and show strongneutralizing activity as assessed with a TF1 cell proliferation assay.

An additional exemplary neutralizing anti-GM-CSF antibody is the MT203antibody described by Krinner et al, (Mol Immunol. 44:916-25, 2007; Epub2006 May 112006). MT203 is an IgG1 class antibody that binds GM-CSF withpicomolar affinity. The antibody shows potent inhibitory activity asassessed by TF-I cell proliferation assay and its ability to block IL-8production in U937 cells.

Additional antibodies suitable for use with the present invention willbe known to persons of skill in the art.

hGM-CSF antagonists that are anti-hGM-CSF receptor antibodies can alsobe employed with the methods of the present disclosure. Such hGM-CSFantagonists include antibodies to the hGM-CSF receptor alpha chain orbeta chain. An anti-hGM-CSF receptor antibody employed in the inventioncan be in any antibody format as explained above, e.g., intact,chimeric, monoclonal, polyclonal, antibody fragment, humanized,Humaneered, and the like. Examples of anti-hGM-CSF receptor antibodies,e.g., neutralizing, high-affinity antibodies, suitable for use in theinvention are known (see, e.g., U.S. Pat. No. 5,747,032 and Nicola etal., Blood 82: 1724, 1993).

Non-Antibody GM-CSF Antagonists

Other proteins that may interfere with the productive interaction ofhGM-CSF with its receptor include mutant hGM-CSF proteins and secretedproteins comprising at least part of the extracellular portion of one orboth of the hGM-CSF receptor chains that bind to hGM-CSF and competewith binding to cell-surface receptor. For example, a soluble hGM-CSFreceptor antagonist can be prepared by fusing the coding region of thesGM-CSFR alpha with the CH2-CH3 regions of murine IgG2a. An exemplarysoluble hGM-CSF receptor is described by Raines et al. (1991) Proc.Natl. Acad. Sci USA 88: 8203. An example of a GM-CSFR alpha-Fc fusionprotein is provided, e.g., in Brown et al (1995) Blood 85: 1488. In someembodiments, the Fc component of such a fusion can be engineered tomodulate binding, e.g., to increase binding, to the Fc receptor.

Other hGM-CSF antagonists include hGM-CSF mutants. For example, hGM-CSFhaving a mutation of amino acid residue 21 of hGM-CSF to Arginine orLysine (E21R or E21K) described by Hercus et al. Proc. Natl. Acad. SciUSA 91:5838, 1994 has been shown to have in vivo activity in preventingdissemination of hGM-CSF-dependent leukemia cells in mouse xenograftmodels (Iversen et al. Blood 90:4910, 1997). As appreciated by one ofskill in the art, such antagonists can include conservatively modifiedvariants of hGM-CSF that have substitutions, such as the substitutionnoted at amino acid residue 21, or hGM-CSF variants that have, e.g.,amino acid analogs to prolong half-life.

In some embodiments, the hGM-CSF antagonist may be a peptide. Forexample, an hGM-CSF peptide antagonist may be a peptide designed tostructurally mimic the positions of specific residues on the B and Chelices of human GM-CSF that are implicated in receptor binding andbioactivity (e.g., Monfardini et al, J. Biol. Chem 271:2966-2971, 1996).

In other embodiments, the hGM-CSF antagonist is an “antibody mimetic”that targets and binds to the antigen in a manner similar to antibodies.Certain of these “antibody mimics” use non-immunoglobulin proteinscaffolds as alternative protein frameworks for the variable regions ofantibodies. For example, Ku et al. (Proc. Natl. Acad. Sci. U.S.A.92(14):6552-6556 (1995)) discloses an alternative to antibodies based oncytochrome b562 in which two of the loops of cytochrome b562 wererandomized and selected for binding against bovine serum albumin. Theindividual mutants were found to bind selectively with BSA similarlywith anti-BSA antibodies. U.S. Pat. Nos. 6,818,418 and 7,115,396disclose an antibody mimic featuring a fibronectin or fibronectin-likeprotein scaffold and at least one variable loop. Known as Adnectins,these fibronectin-based antibody mimics exhibit many of the samecharacteristics of natural or engineered antibodies, including highaffinity and specificity for any targeted ligand. The structure of thesefibronectin-based antibody mimics is similar to the structure of thevariable region of the IgG heavy chain. Therefore, these mimics displayantigen binding properties similar in nature and affinity to those ofnative antibodies. Further, these fibronectin-based antibody mimicsexhibit certain benefits over antibodies and antibody fragments. Forexample, these antibody mimics do not rely on disulfide bonds for nativefold stability, and are, therefore, stable under conditions which wouldnormally break down antibodies. In addition, since the structure ofthese fibronectin-based antibody mimics is similar to that of the IgGheavy chain, the process for loop randomization and shuffling may beemployed in vitro that is similar to the process of affinity maturationof antibodies in vivo.

Beste et al. (Proc. Natl. Acad. Sci. U.S.A. 96(5):1898-1903 (1999))disclose an antibody mimic based on a lipocalin scaffold (Anticalin®).Lipocalins are composed of a β-barrel with four hypervariable loops atthe terminus of the protein. The loops were subjected to randommutagenesis and selected for binding with, for example, fluorescein.Three variants exhibited specific binding with fluorescein, with onevariant showing binding similar to that of an anti-fluorescein antibody.Further analysis revealed that all of the randomized positions arevariable, indicating that Anticalin would be suitable to be used as analternative to antibodies. Thus, Anticalins are small, single chainpeptides, typically between 160 and 180 residues, which provides severaladvantages over antibodies, including decreased cost of production,increased stability in storage and decreased immunological reaction.

U.S. Pat. No. 5,770,380 discloses a synthetic antibody mimetic using therigid, non-peptide organic scaffold of calixarene, attached withmultiple variable peptide loops used as binding sites. The peptide loopsall project from the same side geometrically from the calixarene, withrespect to each other. Because of this geometric confirmation, all ofthe loops are available for binding, increasing the binding affinity toa ligand. However, in comparison to other antibody mimics, thecalixarene-based antibody mimic does not consist exclusively of apeptide, and therefore it is less vulnerable to attack by proteaseenzymes. Neither does the scaffold consist purely of a peptide, DNA orRNA, meaning this antibody mimic is relatively stable in extremeenvironmental conditions and has a long life-span. Further, since thecalixarene-based antibody mimic is relatively small, it is less likelyto produce an immunogenic response.

Murali et al. (Cell MoI Biol 49(2):209-216 (2003)) describe amethodology for reducing antibodies into smaller peptidomimetics, theyterm “antibody-like binding peptidomimetics” (ABiP) which may also beuseful as an alternative to antibodies.

In addition to non-immunoglobulin protein frameworks, antibodyproperties have also been mimicked in compounds comprising RNA moleculesand unnatural oligomers (e.g., protease inhibitors, benzodiazepines,purine derivatives and beta-turn mimics). Accordingly, non-antibodyGM-CSF antagonists can also include such compounds.

Therapeutic Administration

In some embodiments, the methods of the present disclosure compriseadministering a hGM-CSF antagonist, (e.g., an anti-hGM-CSF antibody) asa pharmaceutical composition to a subject having a CRS or a cytokinestorm. In some embodiments, the hGM-CSF antagonist is administered in atherapeutically effective amount using a dosing regimen suitable fortreatment of the disease.

In some embodiments, a therapeutically effective amount is an amountthat at least partially arrests the condition or its symptoms. Forexample, a therapeutically effective amount may arrest immuneactivation, may decrease the levels of circulating cytokines, maydecrease T-cell activation, or may ameliorate fever, malaise, fatigue,anorexia, myalgias, arthalgias, nausea, vomiting, headache, skin rash,nausea, vomiting, diarrhea, tachypnea, hypoxemia, cardiovasculartachycardia, widened pulse pressure, hypotension, increased cardiacoutput (early), potentially diminished cardiac output (late), elevatedD-dimer, hypofibrinogenemia with or without bleeding, azotemia,transaminitis, hyperbilirubinemia, headache, mental status changes,confusion, delirium, word finding difficulty or frank aphasia,hallucinations, tremor, dysmetria, altered gait, or seizures.

The methods of the invention comprise administering an anti-hGM-CSFantibody as a pharmaceutical composition to a patient in atherapeutically effective amount using a dosing regimen suitable fortreatment of the disease. The composition can be formulated for use in avariety of drug delivery systems. One or more physiologically acceptableexcipients or carriers can also be included in the compositions forproper formulation. Suitable formulations for use in the presentinvention are found in Remington: The Science and Practice of Pharmacy,21st Edition, Philadelphia, Pa. Lippincott Williams & Wilkins, 2005. Fora brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990).

The anti-hGM-CSF antibody for use in the methods of the invention isprovided in a solution suitable for injection into the patient such as asterile isotonic aqueous solution for injection. The antibody isdissolved or suspended at a suitable concentration in an acceptablecarrier. In some embodiments the carrier is aqueous, e.g., water,saline, phosphate buffered saline, and the like. The compositions maycontain auxiliary pharmaceutical substances as required to approximatephysiological conditions, such as pH adjusting and buffering agents,tonicity adjusting agents, and the like.

The pharmaceutical compositions of the invention are administered to apatient, e.g., a patient that has osteopenia, rheumatoid arthritis,juvenile idiopathic arthritis, systemic-onset Still's disease, asthma,eosinophilic asthma, eosinophilic esophagitis, multiple sclerosis,psoriasis, atopic dermatitis, plexiform neurofibromatosis, autoimmunelymphoproliferative syndrome (or ALPS, also known as Canale-Smithsyndrome), Ras-associated autoimmune leukoproliferative disorder (orRALD), Noonan syndrome, chronic myelomonocytic leukemia, juvenilemyelomonocytic leukemia, acute myeloid leukemia, MulticentricCastleman's Disease, chronic obstructive pulmonary disease, interstitiallung disease, diffuse parenchymal lung disease, idiopathicthrombocytopenia purpura, Alzheimer's disease, heart failure, Kawasaki'sDisease, cardiac damage due to an ischemic event, or diabetes, in anamount sufficient to cure or at least partially arrest the disease orsymptoms of the disease and its complications. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.” Atherapeutically effective dose is determined by monitoring a patient'sresponse to therapy. Typical benchmarks indicative of a therapeuticallyeffective dose includes amelioration of symptoms of the disease in thepatient. Amounts effective for this use will depend upon the severity ofthe disease and the general state of the patient's health, includingother factors such as age, weight, gender, administration route, etc.Single or multiple administrations of the antibody may be administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the methods provide a sufficient quantity ofanti-hGM-CSF antibody to effectively treat the patient.

The antibody may be administered alone, or in combination with othertherapies to treat the disease of interest.

The antibody can be administered by injection or infusion through anysuitable route including but not limited to intravenous, sub-cutaneous,intramuscular or intraperitoneal routes. In some embodiments, theantibody may be administered by insufflation. In an exemplaryembodiment, the antibody may be stored at 10 mg/ml in sterile isotonicaqueous saline solution for injection at 4° C. and is diluted in either100 ml or 200 ml 0.9% sodium chloride for injection prior toadministration to the patient. The antibody is administered byintravenous infusion over the course of 1 hour at a dose of between 0.2and 10 mg/kg. In other embodiments, the antibody is administered, forexample, by intravenous infusion over a period of between 15 minutes and2 hours. In still other embodiments, the administration procedure is viasub-cutaneous or intramuscular injection.

In some embodiments, the hGM-CSF antagonist, e.g., an anti-hGM-CSFantibody, is administered by a perispinal route. Perispinaladministration involves anatomically localized delivery performed so asto place the therapeutic molecule directly in the vicinity of the spineat the time of initial administration. Perispinal administration isdescribed, e.g., in U.S. Pat. No. 7,214,658 and in Tobinick & Gross, J.Neuroinflammation 5:2, 2008.

The dose of hGM-CSF antagonist is chosen in order to provide effectivetherapy for a subject that has been diagnosed with CRS or cytokinestorm. The dose is typically in the range of about 0.1 mg/kg body weightto about 50 mg/kg body weight or in the range of about 1 mg to about 2 gper patient. The dose is often in the range of about 1 to about 20 mg/kgor approximately about 50 mg to about 2000 mg/patient. The dose may berepeated at an appropriate frequency which may be in the range once perday to once every three months, depending on the pharmacokinetics of theantagonist (e.g. half-life of the antibody in the circulation) and thepharmacodynamic response (e.g. the duration of the therapeutic effect ofthe antibody). In some embodiments where the antagonist is an antibodyor modified antibody fragment, the in vivo half-life of between about 7and about 25 days and antibody dosing is repeated between once per weekand once every 3 months. In other embodiments, the antibody isadministered approximately once per month.

A V_(H) region and/or V_(L) region of the invention may also be used fordiagnostic purposes. For example, the V_(H) and/or V_(L) region may beused for clinical analysis, such as detection of GM-CSF levels in apatient. A V_(H) or V_(L) region of the invention may also be used,e.g., to produce anti-Id antibodies.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of skill in the art. Further, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular.

In one embodiment, “treating” comprises therapeutic treatment and“preventing” comprises prophylactic or preventative measures, whereinthe object is to prevent or lessen the targeted pathologic condition ordisorder as described hereinabove. Thus, in one embodiment, treating mayinclude directly affecting or curing, suppressing, inhibiting,preventing, reducing the severity of, delaying the onset of, reducingsymptoms associated with the disease, disorder or condition, or acombination thereof. Thus, in one embodiment, “treating,”“ameliorating,” and “alleviating” refer inter alia to delayingprogression, expediting remission, inducing remission, augmentingremission, speeding recovery, increasing efficacy of or decreasingresistance to alternative therapeutics, or a combination thereof. In oneembodiment, “preventing” refers, inter alia, to delaying the onset ofsymptoms, preventing relapse to a disease, decreasing the number orfrequency of relapse episodes, increasing latency between symptomaticepisodes, or a combination thereof. In one embodiment, “suppressing” or“inhibiting”, refers inter alia to reducing the severity of symptoms,reducing the severity of an acute episode, reducing the number ofsymptoms, reducing the incidence of disease-related symptoms, reducingthe latency of symptoms, ameliorating symptoms, reducing secondarysymptoms, reducing secondary infections, prolonging patient survival, ora combination thereof.

In the present disclosure the singular forms “a,” “an,” and “the”include the plural reference, and reference to a particular numericalvalue includes at least that particular value, unless the contextclearly indicates otherwise. The term “plurality”, as used herein, meansmore than one. When a range of values is expressed, another embodimentincludes from the one particular and/or to the other particular value.

Similarly, when values are expressed as approximations, by use of theantecedent “about,” it is understood that the particular value formsanother embodiment. All ranges are inclusive and combinable. In someembodiments, the term “about”, refers to a deviance of between 0.0001-5%from the indicated number or range of numbers. In some embodiments, theterm “about”, refers to a deviance of between 1-10% from the indicatednumber or range of numbers. In some embodiments, the term “about”,refers to a deviance of up to 25% from the indicated number or range ofnumbers. The term “comprises” means encompasses all the elements listed,but may also include additional, unnamed elements, and it may be usedinterchangeably with the terms “encompasses”, “includes”, or “contains”having all the same qualities and meanings. The term “consisting of”means being composed of the recited elements or steps, and it may beused interchangeably with the terms “composed of” having all the samequalities and meanings.

EXAMPLES Example 1—Exemplary Humaneered Antibodies to GM-CSF

A panel of engineered Fab′ molecules with the specificity of c19/2 weregenerated from epitope-focused human V-segment libraries as described inUS patent application publication nos. 20060134098 and 20050255552.Epitope-focused libraries were constructed from human V-segment librarysequences linked to a CDR3-FR4 region containing BSD sequences in CDRH3and CDRL3 together with human germ-line J-segment sequences. For theheavy chain, human germ-line JH4 sequence was used and for the lightchain, human germ-line JK4 sequence was used.

Full-length Humaneered V-regions from a Vh1-restricted library wereselected that supported binding to recombinant human GM-CSF. The“full-length” V-kappa library was used as a base for construction of“cassette” libraries as described in US patent application publicationno. 20060134098, in which only part of the murine c19/2 V-segment wasinitially replaced by a library of human sequences. Two types ofcassettes were constructed. Cassettes for the V-kappa chains were madeby bridge PCR with overlapping common sequences within the framework 2region. In this way “front-end” and “middle” human cassette librarieswere constructed for the human V-kappa III isotype. Human V-kappa IIIcassettes which supported binding to GM-CSF were identified bycolony-lift binding assay and ranked according to affinity in ELISA. TheV-kappa human “front-end” and “middle” cassettes were fused together bybridge PCR to reconstruct a fully human V-kappa region that supportedGM-CSF binding activity. The Humaneered Fabs thus consist of HumaneeredV-heavy and V-kappa regions that support binding to human GM-CSF.

Binding activity was determined by surface plasmon resonance (spr)analysis. Biotinylated GM-CSF was captured on a streptavidin-coated CMSbiosensor chip. Humaneered Fab fragments expressed from E. coli werediluted to a starting concentration of 30 nM in 10 mM HEPES, 150 mMNaCl, 0.1 mg/ml BSA and 0.005% P20 at pH 7.4. Each Fab was diluted 4times using a 3-fold dilution series and each concentration was testedtwice at 37 degrees C. to determine the binding kinetics with thedifferent density antigen surfaces. The data from all three surfaceswere fit globally to extract the dissociation constants.

Binding kinetics were analyzed by Biacore 3000 surface plasmon resonance(SPR). Recombinant human GM-CSF antigen was biotinylated and immobilizedon a streptavidin CMS sensor chip. Fab samples were diluted to astarting concentration of 3 nM and run in a 3-fold dilution series.Assays were run in 10 mM HEPES, 150 mM NaCl, 0.1 mg/mL BSA and 0.005%p20 at pH 7.4 and 37° C. Each concentration was tested twice. Fab′binding assays were run on two antigen density surfaces providingduplicate data sets. The mean affinity (KD) for each of 6 varioushumaneered anti-GM-CSF Fab clones, calculated using a 1: 1 Langmuirbinding model, is shown in Table 2.

Fabs were tested for GM-CSF neutralization using a TF-I cellproliferation assay. GM-CSF-dependent proliferation of human TF-I cellswas measured after incubation for 4 days with 0.5 ng/ml GM-CSF using aMTS assay (Cell titer 96, Promega) to determine viable cells. All Fabsinhibited cell proliferation in this assay indicating that these areneutralizing antibodies. There is a good correlation between relativeaffinities of the anti-GM-CSF Fabs and EC50 in the cell-based assay.Anti-GM-CSF antibodies with monovalent affinities in the range 18 pM-104pM demonstrate effective neutralization of GM-CSF in the cell-basedassay.

Exemplary engineered anti-GM-CSF V region sequences are shown in FIG. 1.

TABLE 2 Affinity of anti-GM-CSF Fabs determined by surface plasmonresonance analysis in comparison with activity (EC50) in a GM-CSFdependent TF-I cell proliferation assay Monovalent EC₅₀(pM) in TF-binding affinity 1 cell determined by proliferation Fab SPR (pM) assay94 18 165 104 19 239 77 29 404 92 58 539 42 104 3200 44 81 7000

Example 2—Evaluation of a Humaneered GM-CSF Antibody

This example evaluates the binding activity and biological potency of ahumaneered anti-GM-CSF antibody in a cell-based assay in comparison to achimeric IgG1k antibody (Ab2) having variable regions from the mouseantibody LMM102 (Nice et al., Growth Factors 3:159, 1990). Ab1 is ahumaneered IgG1k antibody against GM-CSF having identical constantregions to Ab2.

Surface Plasmon Resonance Analysis of Binding of Human GM-CSF to Ab1 andAb2

Surface Plasmon resonance analysis was used to compare binding kineticsand monovalent affinities for the interaction of Ab1 and Ab2 withglycosylated human GM-CSF using a Biacore 3000 instrument. Ab1 or Ab2was captured onto the Biacore chip surface using polyclonal anti-humanF(ab′)2. Glycosylated recombinant human GM-CSF expressed from human 293cells was used as the analyte. Kinetic constants were determined in 2independent experiments (see FIGS. 2A-2B and Table 3). The results showthat GM-CSF bound to Ab2 and Ab1 with comparable monovalent affinity inthis experiment. However, Ab1 had a two-fold slower “on-rate” than Ab2,but an “off-rate” that was approximately three-fold slower.

TABLE 3 Kinetic constants at 37° C. determined from the surface plasmonresonance analysis in FIGS. 2A-2B; association constant (k_(a)),dissociation constant (k_(d)) and calculated affinity (KD) are shown.k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) KD (pM) Ab2 7.20 × 10⁵  2.2 × 10⁻⁵ 30.5 Ab12.86 × 10⁵ 7.20 × 10⁻⁶ 25.1

GM-CSF is naturally glycosylated at both N-linked and O-linkedglycosylation sites although glycosylation is not required forbiological activity. In order to determine whether GM-CSF glycosylationaffects the binding of Ab1 or Ab2, the antibodies were compared in anELISA using recombinant GM-CSF from two different sources; GM-CSFexpressed in E. coli (non-glycosylated) and GM-CSF expressed from human293 cells (glycosylated). The results in FIGS. 3A-3B and Table 4 showedthat both antibodies bound glycosylated and non-glycosylated GM-CSF withequivalent activities. The two antibodies also demonstrated comparableEC₅₀ values in this assay.

TABLE 4 Summary of EC₅₀ for binding of Ab2 and Ab1 to human GM-CSF fromtwo different sources determined by ELISA. Binding to recombinant GM-CSF from human 293 cells (glycosylated) or from E. coli (non-glycosylated) was determined from two independent experiments. Experimen1 is shown in FIGS. 3A-3B. Non-glycosylated Non-glycosylatedGlycosylated (exp (exp 1) (exp 2) 1) Ab2 400 pM 433 pM 387 pM Ab1 373 pM440 pM 413 pM

Ab1 is a Humaneered antibody that was derived from the mouse variableregions present in Ab2. Ab1 was tested for overlapping epitopespecificity (Ab2) by competition ELISA.

Biotinylated Ab2 was prepared using known techniques. Biotinylation didnot affect binding of Ab2 to GM-CSF as determined by ELISA. In theassay, Ab2 or Ab1 was added in varying concentrations with a fixedamount of biotinylated Ab2. Detection of biotinylated Ab2 was assayed inthe presence of unlabeled Ab or Ab1 competitor (FIGS. 4A-4B). Both Ab1and Ab2 competed with biotinylated Ab2 for binding to GM-CSF, thusindicating binding to the same epitope. Ab1 competed more effectivelyfor binding to GM-CSF than Ab2, consistent with the slower dissociationkinetics for Ab1 when compared with Ab2 by surface plasmon resonanceanalysis.

Neutralization of GM-CSF Activity by Ab1 and Ab2

A cell-based assay for neutralization of GM-CSF activity was employed toevaluate biological potency. The assay measures IL-8 secretion from U937cells induced with GM-CSF. IL-8 secreted into the culture supernatant isdetermined by ELISA after 16 hours induction with 0.5 ng/ml E.coli-derived GM-CSF.

A comparison of the neutralizing activity of Ab1 and Ab2 in this assayis shown in a representative assay in FIG. 5. In three independentexperiments, Ab1 inhibited GM-CSF activity more effectively than Ab2when comparing IC50 (Table 5).

TABLE 5 Comparison of IC50 for inhibition of GM-CSF induced IL-8expression. Data from three independent experiments shown in FIG. 5 andmean IC₅₀ are expressed in ng/ml and nM. Experiment Ab2 (ng/ml) Ab2 (nM)Ab1 (ng/ml) Ab1 (nM) A 363 2.4 31.3 0.21 B 514 3.4 92.5 0.62 C 343 2.220.7 0.14 Mean 407 2.7 48.2 0.32

Summary

The Humaneered Ab1 bound to GM-CSF with a calculated equilibrium bindingconstant (KD) of 25 pM. Ab2 bound to GM-CSF with a KD of 30.5 pM. Ab2showed a two-fold higher association constant (k_(a)) than Ab1 forGM-CSF while Ab1 showed three-fold slower dissociation kinetics (k_(d))than Ab2. Ab2 and Ab1 showed similar binding activity for glycosylatedand non-glycosylated GM-CSF in an antigen-binding ELISA. A competitionELISA confirmed that both antibodies competed for the same epitope; Ab1showed higher competitive binding activity than Ab2. In addition, Ab1showed higher GM-CSF neutralization activity than Ab2 in aGM-CSF-induced IL-8 induction assay.

Example 3—Administration of a Neutralizing Anti-GM-CSF Antibody in aMouse Model of Immunotherapy-Related Toxicity

A mouse model of immunotherapy-related toxicity can be used to show theefficacy of an anti-GM-CSF antibody for preventing and treatingimmunotherapy-related toxicity. In one model of immunotherapy-relatedtoxicity, mice are injected with CAR T-cells in doses provokingtoxicity. For example, van der Stegen et al. (J. Immunol 191:4589-4598(2013)), incorporated herein by reference, describe a CRS model inducedby the i.p. injection of a single dose of 30×10⁶ cells termed T4⁺ Tcells. T4⁺ T cells are engineered T cells expressing the chimeric Agreceptor (CAR) T1E28z. T cells engineered to express T1E28z areactivated by cells expressing ErbB1- and ErbB4-based dimers and ErbB2/3heterodimer.

To evaluate the efficacy of anti-GM-CSF antibodies for preventing andtreating CRS, mice will be divided in groups (n=10), each groupreceiving either: a) a single i.p. saline injection; b) an i.p.injection of 30×10⁶ T4⁺ T cells; c) an i.p. injection of 30×10⁶ T4⁺ Tcells and 0.25 mg intravenous (i.v.) anti-GM-CSF monoclonal antibody22E9 (a recombinant rat anti-mouse-GM-CSF antibody) co-administered withT4⁺ T cells; d) an i.p. injection of 30×10⁶ T4⁺ T cells and 0.25 mgintranasal (i.n.) anti-GM-CSF antibody 22E9 co-administered with T4⁺ Tcells; e) an i.p. injection of 30×10⁶ T4⁺ T cells and 0.25 mg i.v.anti-GM-CSF antibody 22E9 6 hours before T4⁺ T cells administration; f)an i.p. injection of 30×10⁶ T4⁺ T cells and 0.25 mg i.n. anti-GM-CSFantibody 22E9 6 hours before T4⁺ T cells administration; g) an i.p.injection of 30×10⁶ T4⁺ T cells and 0.25 mg i.v. anti-GM-CSF antibody22E9 2 hours after T4⁺ T cells administration; or h) an i.p. injectionof 30×10⁶ T4⁺ T cells and 0.25 mg i.n. anti-GM-CSF antibody 22E9 2 hoursafter T4⁺ T cells administration. Further doses, administration times,and administration routes will be evaluated.

In order to assess anti-GM-CSF antibody 22E9 effect, organs will becollected from mice, formalin fixed, and subjected to histopathologicanalysis. Blood will be collected and concentrations of human IFNγ,human IL-2, and mouse IL-6, IL-2, IL-4, IL-6, IL-10, IL-17, IFNγ, andTNFα will be assessed by well methods described in the literature, suchas ELISA assay. Mice weight, behavior, and clinical manifestations willbe observed.

Example 4—Anti-GM-CSF Antibody Effect on Immunotherapy

A mouse model can be used to show that GM-CSF antagonists do notnegatively affect the efficacy of cancer immunotherapy. SCID beige micecan be inoculated with a cancer cell line and treated with animmunotherapeutic agent known to induce CRS, as T4⁺ T cells, with orwithout an anti-GM-CSF antibody.

To evaluate whether anti-GM-CSF antibodies affect the efficacy ofimmunotherapy, mice will be divided in groups (n=10), each groupreceiving either: a) a subcutaneous (s.c.) injection of 30×10⁶ SKOV3cells; b) a s.c. injection of 30×10⁶ SKOV3 cells and an i.p. injectionof 30×10⁶ T4⁺ T cells; or c) a s.c. implant of 30×10⁶ SKOV3 cells, ani.p. injection of 30×10⁶ T4⁺ T cells, and an i.v. injection of 0.25 mgof anti-GM-CSF antibody 22E9.

In order to assess anti-GM-CSF antibody 22E9 effect on T4⁺ T cellsefficacy, tumor size will be measured every four days by caliper, andtumor volume calculated by the formula: 0.5×(larger diameter)×(smallerdiameter)². Mice weight, behavior, and clinical manifestations will beobserved. At the end of the experiment, the animals will be sacrificed,and the tumor tissues harvested and weighted.

Example 5—Mouse Model of Human CRS

A mouse model for CRS for investigating the effects of a humanizedanti-GM-CSF monoclonal antibody in treating or preventing CRS wasdeveloped. (FIGS. 17A-17B).

Method: The model used is a primary AML model. Immunocompromised NSG-Smice that were additionally transgenic for human SCF, IL-3, and GM-CSFwere engrafted with AML blasts derived from AML patients that were CD123positive. After 2-4 weeks, they were bled to confirm engraftment andachievement of high disease burden. The mice were then treated with highdoses of CAR-T123 at 1×10⁶ cells, which is 10 times higher than dosespreviously studied.

Results: It was observed that within 1-2 weeks after CAR-T cellinjection, these mice developed an illness characterized by weakness,emaciation, hunched bodies, withdrawal, and poor motor response. Themice eventually died of their disease within 7-10 days. The symptomscorrelate with massive T-cell expansion in the mice and with elevationof multiple human cytokines, such as IL-6, MIP 1α, IFN-γ, TNFα, GM-CSF,MIP1β, and IL-2, and in a pattern that resembles what is seen in humanCRS after CAR-T cell therapy. GM-CSF fold change was significantlygreater than other cytokines. (FIGS. 17A-17B).

Example 6—Generation of GM-CSF Knockout CAR-Ts

GM-CSF CRISPR knockout T cells were generated and shown to exhibitreduced expression of GM-CSF but similar levels of other cytokines anddegranulation, which showed immune cell functionality. (See FIGS.15A(i), 15A(ii), 15B-15G).

Example 7—Anti-GM-CSF Neutralizing Antibody does not Inhibit CAR-TMediated Killing, Proliferation, or Cytokine Production but NeutralizesGM-CSF

Anti-GM-CSF neutralizing antibody does not inhibit CAR-T mediatedkilling, proliferation, or cytokine production but successfullyneutralizes GM-CSF. (See FIGS. 16A(i), 16A(ii), 16B-16J).

Example 8—Anti-GM-CSF Neutralizing Antibody does not Inhibit CAR-TEfficacy In Vivo

Humanized anti-GM-CSF monoclonal antibody, a neutralizing hGM-CSFantibody, does not inhibit CAR-T efficacy in vivo (FIGS. 18A-18C). CAR-Tefficacy in a xenograft model in combination with an anti-GM-CSFneutralizing antibody in accordance with embodiments described herein.As shown in FIG. 18A, NSG mice were injected with NALM-6-GFP/Luciferasecells (human, peripheral blood leukemia pre-B cell), and bioluminescentimaging (BLI0 was performed to confirm tumor growth. Mice were treatedwith either (1) anti-GM-CSF antibody (10 mg/Kg daily for ten days) and(a) CART19 or (b) untransduced human T cells (UTD) 1×10⁶ cells or (2)IgG control antibody (10 mg/Kg daily for ten days) and (a) CART19 or (b)untransduced human T cells (UTD) 1×10⁶ cells. FIGS. 18B and 18Cdemonstrate that the anti-GM-CSF neutralizing antibody did not inhibitCAR-T efficacy in vivo.

Example 9—Anti-GM-CSF Neutralizing Antibody does not Impair CAR-T Impacton Survival

In vitro and in vivo preclinical data show anti-GM-CSF neutralizingantibody (a humanized anti-GM-CSF monoclonal antibody) does not impairCAR-T impact on survival in mouse models. (FIG. 19).

The anti-GM-CSF neutralizing antibody does not impede CAR-T cellfunction in vivo in the absence of PBMCs. Survival shown to be similarfor CAR-T+control and CAR-T+anti-GM-CSF neutralizing antibody.

Example 10—Anti-GM-CSF Neutralizing Antibody May Increase CAR-TExpansion

In vitro and In vivo preclinical data show anti-GM-CSF neutralizingantibody (a humanized anti-GM-CSF monoclonal antibody) may increaseCAR-T Expansion (FIGS. 20A-20B). The anti-GM-CSF neutralizing antibodymay increase in vitro CAR-T cancer cell killing. The antibody increasesproliferation of CAR-T cells and could improve efficacy. CAR-Tproliferation increased by the GM-CSF neutralizing antibody in presenceof PBMCs. (It was not affected without PBMCs). The antibody did notinhibit degranulation, intracellular GM-CSF production, or IL-2production.

Example 11—CAR-T Expansion Associated with Improved Overall ResponseRate

CAR-T expansion associated with improved overall response rate. (FIG.21). CAR AUC (area under the curve) defined as cumulative levels ofCAR+cells/μL of blood over the first 28 days post CAR-T administration.P values calculated by Wilcoxon rank sum test. (Neelapu, et al ICML 2017Abstract 8).

Example 12—Study Protocol for an Anti-GM-CSF Neutralizing Antibody inAccordance with Embodiments Described Herein

Study protocol for an anti-GM-CSF neutralizing antibody (a humanizedanti-GM-CSF monoclonal antibody) in accordance with embodimentsdescribed herein. (See FIG. 22). CRS and NT to be assessed daily whilehospitalized and at clinic visit for first 30 days. Eligible subjects toreceive GM-CSF neutralizing antibody on days −1, +1, and +3 of CAR-Ttreatment. Tumor assessment to be performed at baseline and months 1, 3,6, 9, 12, 18, and 24. Blood samples (PBMC and serum) days −5, −1, 0, 1,3, 5, 7, 9, 11, 13, 21, 28, 90, 180, 270, and 360.

Example 13—GM-CSF Depletion Increases CAR-T Cell Expansion

GM-CSF depletion increases CAR-T cell expansion. (FIG. 23A-23B) FIG. 23Ashows increased ex-vivo expansion of GM-CSF^(k/o) CAR-T cells comparedto control CAR-T cells. FIG. 23B demonstrates more robust proliferationafter in vivo treatment with an anti-GM-CSF neutralizing antibody (ahumanized anti-GM-CSF monoclonal antibody) in accordance withembodiments described herein.

Example 14—Safety Profile of an Anti-GM-CSF Neutralizing Ab in >100Human Patients* Phase I:

Single-dose, dose escalation in healthy adult volunteers. Objectiveswere to analyze

Safety/tolerability, PK, and Immunogenicity.

Enrollment/dose:

(n=12)

3/1 mg/kg

3/3 mg/kg

3/10 mg/kg

3/placebo

Safety Results:

-   -   Clean Safety Profile:    -   No drug related serious adverse effects (SAE)    -   Non-immunogenic

Phase II:

1) Dose at weeks 0, 2, 4, 8, 12 in rheumatoid arthritis patients.Objectives were to analyze Efficacy, Safety/tolerability, PK, andImmunogenicity.

Enrollment/dose:

(n=9)

7/600 mg

2/placebo

Safety Results:

-   -   Clean Safety Profile:    -   No drug related serious adverse effects (SAE)    -   Non-immunogenic

2) Dose at weeks 0, 2, 4, 8, 12, 16 20 in severe asthma patients.Objectives were to analyze Efficacy, Safety/tolerability, PK, andImmunogenicity.

Enrollment/dose:

(n=160)

78/400 mg

82/placebo

Safety Results:

-   -   Clean Safety Profile:    -   No drug related serious adverse effects (SAE)    -   Non-immunogenic        *94 patients in studies depicted above, plus 12 patients in        ongoing CMML Phase I trial, where drug is well tolerated; an        additional 76 patients received a chimeric version of a GM-CSF        neutralizing Ab (KB002) and showed a similar safety profile.

All studies randomized double-blind placebo-controlled, IVadministration. (See FIG. 24.)

Example 15—Effect of Anti-GM-CSF Antibody on CART Activity and Toxicity

The study will investigate the effect of GMCSF blockade with anti-GM-CSFantibody on chimeric antigen receptor T cells (CART) activity andtoxicity. This can be accomplished through these two AIMS:

AIM#1: to investigate the effect of GMCSF blockade with anti-GM-CSFantibody on CART cell effector functionsAIM#2: To study the effect of GMCSF blockade with anti-GM-CSF antibodyon reducing cytokine release syndrome after CART cell therapy Researchstrategy. The following experiments are proposed:

In vitro studies of the combination of four different doses of GMCSFblockade with anti-GM-CSF antibody with CART cells (cytokine production(30 plex Lumiex, including GM-CSF, IL-2, INFg, IL-6, IL-8, MCP-1),antigen specific killing, degranulation, proliferation and exhaustion),in the presence or absence of myeloid cells using the model: CART19against ALL.

In vivo studies of the combination of different doses of GMCSF blockadewith anti-GM-CSF antibody (with and without murine GMCSF blockade) withCART cells, using two models:

CD19 positive cell line (NALM6) engrafted xenografts, treated withCART19 with or without anti-GM-CSF antibody; andPatient derived xenografts with primary ALL, and then treated withCART19 with or without anti-GM-CSF antibody.

Mice will be dosed i.p with anti-GM-CSF antibody 10 mg/kg immediatelyprior to CART cell implantation and 10 mg/kg/day for 10 days. Mice willbe followed for tumor response and survival. Retro-orbital bleedingswill be obtained starting one week after CART cell therapy and weeklyafterwards. Disease burden, T cell expansion kinetics, expression ofexhaustion markers and cytokine levels (30 Plex) will be analyzed. Atthe completion of the experiment, spleens and bone marrows will beharvested and analyzed for tumor characteristics and CAR-T cell numbers.

In vivo studies of the combination of GMCSF blockade with anti-GM-CSFantibody (with or without murine GMCSF blockade) with CART cells in CRSmodels (in this model, high doses of CART cells will be used to elicitCRS), in the presence of PBMCs, using the following model:

Primary ALL patient derived xenografts, then treated with CART19 with orwithout anti-GM-CSF antibody.

Mice will be dosed i.p with anti-GM-CSF antibody 10 mg/kg immediatelyprior to CART cell implantation and 10 mg/kg/day for 10 days. Mice willbe followed for tumor response, CRS toxicity symptoms and survival.Retro-orbital bleedings will be obtained at baseline, 2 days post, oneweek-post CART cell therapy and weekly afterwards. Disease burden, Tcell expansion kinetics, expression of exhaustion markers and cytokinelevels (30 Plex) will be analyzed. At the completion of the experiment,spleens and bone marrows will be harvested and analyzed for tumorcharacteristics and CAR-T cell numbers

In Vivo Neurotoxicity Assays

Using models discussed in #3 above, mice will be imaged with MRI whilesick to assess for development of neurotoxicity after CART cell therapy.Images will be compared between mice that received CART cells andanti-GM-CSF antibody vs control antibody. Repeat experiments will beperformed. Mice will be euthanized 14 days after CART cells in theserepeat experiments. Brain tissue will be analyzed for cytokines withmultiplex assays, for the presence of monocytes, human T cells, and forintegrity of blood brain barrier by IHC, flow and microscopy.

Example 16 Anti-hGM-CSF Neutralizing Antibody Reduces Neuroinflammationin CAR-T Cell Related Neurotoxicity (NT)

There is extensive scientific rationale implicating GM-CSF as essentialto the initiation of cytokine release syndrome (CRS), neurotoxicity (NT)and the inflammatory cascade seen following initiation of CAR-T celltherapy. The hypothesis studied is that blocking soluble GM-CSF with theneutralizing antibody (lenzilumab) will abrogate or prevent the onsetand severity of both CRS and NT observed with CAR-T cell therapy.Importantly, CAR-T cell activity should be preserved or improved ifpossible. The experimental design tests the effects of GM-CSF blockadewith anti-GM-CSF antibody (lenzilumab) on CAR-T cell effector functions,CAR-T efficacy in a tumor xenograft model, development of CRS in a CRSxenograft model and the development of NT using MRI imaging andvolumetric analysis to quantify the neuro-inflammation seen with CAR-Tcell therapy. In vitro and in vivo experiments with CAR-T+/−lenzilumabboth in the presence and absence of human PBMCs were studied. (seeExamples 9 and 10, FIGS. 19 and 20A-20B).

Methods

In vitro studies were conducted to evaluate the combination of GM-CSFneutralizing antibody lenzilumab with human CD19+ CAR-T cells onantigen-specific killing, degranulation, proliferation and exhaustion inthe presence or absence of human PBMCs.

To assess the impact of anti-GM-CSF antibody (lenzilumab) on CAR-T cellproliferation and efficacy, in vivo studies were subsequently conductedusing the following model (with and without murine GM-CSF blockade):

Effector/Target Control Experiments:

CD19 positive cell line (NALM6) engrafted xenografts, treated withCART19 with or without anti-GM-CSF antibody (lenzilumab) in the absenceof human PBMCs.

NSG mice were dosed i.p. with anti-GM-CSF antibody (lenzilumab) 10 mg/kgimmediately prior to CAR-T cell implantation and at the same dose everyday thereafter for 10 days and followed to assess tumor response andsurvival. Retro-orbital bleedings were obtained starting one week afterCAR-T cell therapy and weekly afterwards. Disease burden, T cellexpansion kinetics, expression of exhaustion markers and cytokine levels(30 Plex) were also analyzed. At the completion of the experiment,spleens and bone marrows were harvested and analyzed for tumorcharacteristics and CAR-T cell numbers.

CRS/NT Experiments: Patient Derived Xenografts with Primary ALL,Subsequently Treated with CART19 with or without Lenzilumab in thePresence of Human PBMCs:

To assess the impact of lenzilumab on abrogating or preventing the onsetand severity of CAR-T induced CRS and NT, in vivo studies were conductedwith human CAR-T cells (with and without murine GM-CSF blockade) in aCRS model (where high doses of CAR-T cells were used to illicit CRS) inthe presence of PBMCs using primary ALL patient derived xenografts,treated with CART19 with and without lenzilumab. NSG mice were dosed i.pwith lenzilumab 10 mg/kg immediately prior to CAR-T cell implantationand every day thereafter for 10 days. Mice were followed for tumorresponse, survival, CRS and NT symptoms. Brain MRI scans were taken atbaseline, during and at the end of CAR-T cell therapy and volumetricanalysis was conducted to assess and quantify neuro-inflammation and MRIT2 FLAIR across treatment arms. Body weight and retro-orbital bleedingswere obtained at baseline, 2 days post, one week-post CAR-T cell therapyand weekly afterwards. Disease burden, T cell expansion kinetics,expression of exhaustion markers and cytokine levels (30 Plex) wereanalyzed. At the completion of the experiment, spleens and bone marrowswere harvested and analyzed for tumor characteristics and CAR-T cellnumbers.

Results In Vitro Model

In this experiment, the impact of GM-CSF neutralization with lenzilumabon CAR-T cell effector functions was investigated. It was demonstratedthat GM-CSF is secreted by CAR-T cells at very high levels (over 1,500pg/ml) and the use of lenzilumab completely neutralized GM-CSF but didnot inhibit CAR-T degranulation, intracellular GM-CSF production or IL2production. Moreover, lenzilumab did not inhibit CAR-T antigen specificproliferation or CAR-T killing. Effector-to-target rations (E:T) weresimilar with CAR-T+lenzilumab vs. CAR-T+control antibody, p=ns (FIGS.16A-16D and 16J).

In Vivo Models: Effector/Target Control Experiments:

To study the effect of lenzilumab on CART19 cell function in vivo, weengrafted immuno-compromised NOD-SCID-g−/− with the CD19+ ALL cell lineNALM6 in the absence of human PBMCs. Treatment with CART19 combined withlenzilumab resulted in potent anti-tumor activity and improved overallsurvival, similar to CART19 with control antibody despite completeneutralization of GM-CSF levels in these mice, indicating that GM-CSFdoes not impair CAR-T cell activity in vivo in the absence of PBMCs(FIGS. 16F and 16G).

CRS and NT Experiments:

Using human ALL blasts, human CD19 CAR-T, and human PBMCs, lenzilumab incombination with CAR-T cell therapy was found to reduceneuro-inflammation by ˜90% compared to CAR-T alone as assessed byquantitative MRI T2 FLAIR. This is a landmark finding and the first timeit has been demonstrated in vivo that the neuroinflammation caused byCAR-T cell therapy can be effectively abrogated. MRI images followinglenzilumab plus CAR-T cell therapy were similar to baselinepre-treatment scans, in sharp contrast to MRI images following controlantibody plus CAR-T cell therapy which showed marked increasedinflammation. Moreover, a decrease in myeloid cells was seen in thebrains of mice treated with lenzilumab plus CAR-T compared to micetreated with CAR-T and control antibody. This finding is consistent withdata reported in clinical trials with CD19 CAR-T cell therapy where anincrease in myeloid cells was observed in the CSF of patients withsevere grade>3 neurotoxicity. In addition, lenzilumab in combinationwith CAR-T cell therapy was found to reduce the onset and severity ofCRS as compared to CAR-T plus control antibody. This finding issupported by the statistically significant reduction in body weight seenin mice treated with CAR-T plus control, the most objective marker andhallmark symptom of CRS seen in vivo. In mice treated with lenzilumabplus CAR-T, body weight was maintained at baseline levels as compared toCAR-T plus control (p<0.05). Moreover, mice treated with CAR-T pluscontrol antibody displayed physical symptoms consistent with CRSincluding hunched posture, withdrawal, and weakness while mice treatedwith CAR-T plus lenzilumab appeared healthy. Importantly, lenzilumabplus CAR-T also demonstrates a significant 5-fold increase in theproliferation of CAR-T cells compared to CAR-T plus control in theseCRS/NT experiments that included PBMCs. It has been previously shown inclinical trials with various CD19 CAR-T cell therapies that improvedCAR-T proliferation or expansion correlates with improved efficacy(including ORR, CR), suggesting that lenzilumab may potentially improveanti-tumor response. This finding may be in part explained by a decreasein MDSC expansion and trafficking which is known to be promulgated byGM-CSF. Lastly, the combination of lenzilumab plus CAR-T results insignificantly better leukemic control as quantified by flow cytometrycompared to CAR-T and control antibody. Compared to untreated mice(which had 500,000 to 1.5M leukemic cells) and CAR-T plus controlantibody (which had between 15,000 and 100,000 leukemic cells),treatment with CAR-T plus lenzilumab led to a significant reduction inthe number of leukemic cells (decreased to between 500 and 5,000 cells)with improved overall disease control (see FIGS. 25A-25D).

The MRI images in FIG. 25A shows a clear improvement in neurotoxicity(NT) (neuroinflammation) in the brains of mice administered CAR-T cellsand anti-GM-CSF neutralizing antibody in accordance with embodimentsdescribed herein. In contrast, the brains of mice administered CAR-Tcells and a control antibody showed signs of neurotoxicity in the MRIimages. FIG. 25B graphically illustrates that the NT was reduced by 90%in the mice of Group 1 compared to the NT increased in Group 2 mice. Theextent of quantitative improvement (90% reduction in NT) afteradministration of CAR-T cells and anti-GM-CSF neutralizing antibody inaccordance with embodiments described herein was an unexpected finding.

Conclusions

Anti-GM-CSF antibody (Lenzilumab), when combined with CAR-T cell therapydemonstrates the potential to prevent the onset and severity of CRS andNT, while improving CAR-T expansion/proliferation and overall leukemiccontrol in-vivo using human ALL blasts, human CD19 CAR-T and humanPBMCs. This is the first time it has been demonstrated that CAR-Tinduced neurotoxicity can be abrogated in-vivo. Pivotal clinical trialswith lenzilumab in combination with CAR-T cell therapy are planned tovalidate these findings of improved safety and efficacy.

Example 17 GM-CSF Blockade During Chimeric Antigen Receptor T CellTherapy Reduces Cytokine Release Syndrome and Neurotoxicity and MayEnhance their Effector Functions

Despite its efficacy, chimeric antigen receptor T-cell therapy (CART) islimited by the development of cytokine release syndrome (CRS) andneurotoxicity (NT). While CRS is related to extreme elevation ofcytokines and massive T cell expansion, the exact mechanisms for NT havenot yet been elucidated. Preliminary studies suggest that NT might bemediated by myeloid cells that cross the blood brain barrier. This issupported by correlative analysis from CART19 pivotal trials where CD14+cell numbers were increased in the cerebrospinal fluid of patients thatdeveloped severe NT (Locke et al, ASH 2017). Therefore, the aimed ofthis study was to investigate the role of GM-CSF neutralization inpreventing CRS and NT after CART cell therapy via monocyte control.

First, the effect of GM-CSF blockade on CART cell effector functions wasinvestigated. Here, the human GM-CSF neutralizing antibody (lenzilumab,Humanigen, Burlingame, Calif.) was used that has been shown to be safein phase II clinical trials. Lenzilumab (10 ug/kg) neutralizes GM-CSFwhen CART19 cells are stimulated with the CD19+ Luciferase+ acutelymphoblastic leukemia (ALL) cell line NALM6, but does not impair CARTcell function in vitro. It was found that malignancy associatedmacrophages reduce CART proliferation. GM-CSF neutralization withlenzilumab results in enhanced CART cell antigen specific proliferationin the presence of monocytes. To confirm this in vivo, NOD-SCID-g−/−mice were engrafted with high disease burdens of NALM6 and treated withlow doses of CART19 or control T cells (to induce tumor relapse), incombination with lenzilumab or isotype control antibody. The combinationof CART19 and lenzilumab resulted in significant anti-tumor activity andoverall survival benefit compared to control T cells (FIG. 26A), similarto mice treated with CART19 combined with isotype control antibody,indicating that GM-CSF neutralization does not impair CART cell activityin vivo. This anti-tumor activity was validated in an ALL patientderived xenograft model.

Next, explored was the impact of GM-CSF neutralization on CART cellrelated toxicities in a novel patient derived xenograft model. Here,NOD-SCID-g−/− mice were engrafted with leukemic blasts (1-3×106 cells)derived from patients with high risk relapsed ALL. Mice were thentreated with high doses of CART19 cells (2-5×106 intravenously). Fivedays after CART19 treatment, mice began to develop progressive motorweakness, hunched bodies, and weight loss that correlated with massiveelevation of circulating human cytokine levels. Magnetic ResonanceImaging (MRI) of the brain during this syndrome showed diffuseenhancement and edema, associated with central nervous system (CNS)infiltration of CART cells and murine activated myeloid cells. This issimilar to what has been reported in CART19 clinical trials in patientswith severe NT. The combination of CART19, lenzilumab (to neutralizehuman GM-CS) and murine GM-CSF blocking antibody (to neutralize mouseGM-CSF) resulted in prevention of weight loss (FIG. 26B), decrease incritical myeloid cytokines (FIGS. 26C-26D), reduction of cerebral edema(FIG. 26E), enhanced leukemic disease control in the brain (FIG. 26F),and reduction in brain macrophages (FIG. 26G).

Finally, it was hypothesized that disrupting GM-CSF through CRISPR/Cas9gene editing during the process of CART cell manufacture would result infunctional CART cells with reduced secretion of GM-CSF. Guide RNAtargeting exon 3 of the GM-CSF gene was designed and GM-CSF^(k/o) CART19cells were generated. The preliminary data suggest that these CARTsproduce significantly less GM-CSF upon activation but continue toexhibit similar production of other cytokines and exhibit normaleffector functions in vitro (FIG. 26H). Using the NALM6 high tumorburden relapse xenograft model as described above, GM-CSF^(k/o) CART19cells resulted in slightly enhanced disease control compared to CART19cells (FIG. 26I).

Thus, modulating myeloid cell behavior through GM-CSF blockade can helpcontrol CART mediated toxicities and may reduce their immunosuppressivefeatures to improve leukemic control. These studies illuminate a novelapproach to abrogate NT and CRS through GM-CSF neutralization that alsopotentially enhances CART cell functions. Based on these results, aphase II clinical trial has been designed using lenzilumab as a modalityto prevent CART related toxicities in patients with diffuse large B celllymphoma.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

All publications, accession numbers, patents, and patent applicationscited in this specification are herein incorporated by reference as ifeach was specifically and individually indicated to be incorporated byreference.

Exemplary V_(H) Region Sequences of Anti-GM-CSF Antibodies of theInvention:

(VH#1, FIG. 1) SEQ ID NO: 1QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRRD RFPYYFDYWGQGTLVTVSS(VH#2, FIG. 1) SEQ ID NO: 2QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCARRD RFPYYFDYWGQGTLVTVSS(VH#3, FIG. 1) SEQ ID NO: 3QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCARRQ RFPYYFDYWGQGTLVTVSS(VH#4, FIG. 1) SEQ ID NO: 4QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCVRRQ RFPYYFDYWGQGTLVTVSS(VH#5, FIG. 1) SEQ ID NO: 5QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCVRRQ RFPYYFDYWGQGTLVTVSS

Exemplary V_(L) Region Sequences of Anti-GM-CSF Antibodies of theInvention:

SEQ ID NO: 6 (VK#1, FIG. 1)EIVLTQSPATLSVSPGERATLSCRASQSVGTNVAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNRSPLTFGG GTKVEIKSEQ ID NO: 7 (VK#2, FIG. 1)EIVLTQSPATLSVSPGERATLSCRASQSVGTNVAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNKSPLTFGG GTKVEIKSEQ ID NO: 8 (VK#3, FIG. 1)EIVLTQSPATLSVSPGERATLSCRASQSIGSNLAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNRSPLTFGG GTKVEIKSEQ ID NO: 9 (VK#4, FIG. 1)EIVLTQSPATLSVSPGERATLSCRASQSIGSNLAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNKSPLTFGG GTKVEIKSEQ ID NO: 10 Exemplary kappa constant regionRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECSEQ ID NO: 11 Exemplary heavy chain constant region, f-allotype:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1. A method of inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity in a subject, the method comprising astep of administering a recombinant hGM-CSF antagonist to the subject,wherein said administering inhibits or reduces the incidence or theseverity of immunotherapy-related toxicity in said subject.
 2. Themethod of claim 1, wherein said immunotherapy comprises adoptive celltransfer, administration of monoclonal antibodies, administration ofcytokines, administration of a cancer vaccine, T cell engagingtherapies, or any combination thereof.
 3. The method of claim 2, whereinsaid adoptive cell transfer comprises administering chimeric antigenreceptor-expressing T-cells (CAR T-cells), T-cell receptor (TCR)modified T-cells, tumor-infiltrating lymphocytes (TIL), chimeric antigenreceptor (CAR)-modified natural killer cells, or dendritic cells, or anycombination thereof.
 4. The method of claim 2, wherein said monoclonalantibody is selected from a group comprising: anti-CD3, anti-CD52,anti-PD1, anti-PD-L1, anti-CTLA4, anti-CD20, anti-BCMA antibodies,bi-specific antibodies, or bispecific T-cell engager (BiTE) antibodies,or any combination thereof.
 5. The method of claim 2, wherein saidcytokines are selected from a group comprising: IFNα, IFNβ, IFNγ, IFNλ,IL-2, IL-7, IL-15, IL-21, IL-11, IL-12, IL-18, hGM-CSF, TNFα, or anycombination thereof.
 6. The method of claim 1, wherein said inhibitingor reducing the incidence or the severity of immunotherapy-relatedtoxicity comprises reducing the concentration of at least oneinflammation-associated factor in the serum, tissue fluid, or in the CSFof said subject.
 7. The method of claim 6, wherein saidinflammation-associated factor is selected from a group comprising:C-reactive protein, hGM-CSF, IL-2, sIL2Rα, IL-5, IL-6, IL-8, IP10,IL-10, IL-15, MCP-1, MIG, MIP1β, IFNγ, CX3CR1, or TNFα, or anycombination thereof.
 8. The method of claim 1, wherein administration ofsaid recombinant hGM-CSF antagonist does not reduce the efficacy of saidimmunotherapy.
 9. The method of claim 1, wherein said immunotherapy isadministered at higher doses that it would be administered without theadministration of said hGM-CSF antagonist.
 10. The method of claim 1,wherein administration of said recombinant hGM-CSF antagonist occursprior to, concurrent with, or following said immunotherapy.
 11. Themethod of claim 1, wherein said recombinant hGM-CSF antagonist isco-administered with corticosteroids, anti-IL-6 antibodies, tocilizumab,cyclosporine, antiepileptics, benzodiazepines, acetazolamide,hyperventilation therapy, or hyperosmolar therapy, or any combinationthereof.
 12. The method of claim 1, wherein said immunotherapy-relatedtoxicity comprises a brain disease, damage or malfunction.
 13. Themethod of claim 12, wherein said brain disease, damage or malfunctioncomprises CAR-T cell related neurotoxicity or CAR-T cell relatedencephalopathy syndrome (CRES).
 14. The method of claim 13, wherein theCAR-T cell related neurotoxicity is reduced by about 90% compared to areduction in neurotoxicity in a subject treated with CAR-T cells and acontrol antibody.
 15. The method of claim 1, wherein said inhibiting orreducing incidence of a brain disease, damage or malfunction comprisesreducing headaches, delirium, anxiety, tremor, seizure activity,confusion, alterations in wakefulness, hallucinations, dysphasia,ataxia, apraxia, facial nerve palsy, motor weakness, seizures,nonconvulsive EEG seizures, altered levels of consciousness, coma,endothelial activation, vascular leak, intravascular coagulation, or anycombination thereof in said subject.
 16. The method of claim 12, whereinthe serum concentration of ANG2 or VWF, or the serum ANG2:ANG1 ratio ofsaid subject is reduced.
 17. The method of claim 12, wherein saidsubject has a body temperature above 38° C., IL6 serum concentrationabove 16 pg/ml, or MCP-1 serum concentration above 1,300 pg/ml duringthe first 36 hours after infusion of said CAR-T cells.
 18. The method ofclaim 12, wherein said subject is predisposed to have said braindisease, damage or malfunction.
 19. The method of claim 12, wherein saidsubject has an ANG2:ANG1 ratio in serum above 1 prior to the infusion ofsaid CAR-T cells.
 20. The method of claim 1, wherein saidimmunotherapy-related toxicity comprises hemophagocyticlymphohistiocytosis (HLH) or macrophage-activation syndrome (MAS). 21.The method of claim 20, wherein said inhibiting or reducing incidence ofHLH or MAS comprises increasing survival time and/or time to relapse,reducing macrophage activation, reducing T cell activation, reducing theconcentration of circulating IFNγ, or reducing the concentration ofcirculating of hGM-CSF, or any combination thereof.
 22. The method ofclaim 20, wherein said subject presents with fever, splenomegaly,cytopenias involving two or more lines, hypertriglyceridemia,hypofibrinogenemia, hemophagocytosis, low or absent NK-cell activity,ferritin serum concentration above 500 U/ml, or soluble CD25 serumconcentration above 2400 U/ml, or any combination thereof.
 23. Themethod of claim 20, wherein said subject is predisposed to acquiring HLHor MAS.
 24. The method of claim 20, wherein said subject carries amutation in a gene selected from: PRF1, UNC13D, STX11, STXBP2, orRAB27A, or has reduced expression of perforin, or any combinationthereof.
 25. The method of claim 1, wherein the hGM-CSF antagonist is ananti-hGM-CSF antibody.
 26. The method of claim 25, wherein theanti-hGM-CSF antibody blocks binding of hGM-CSF to the alpha subunit ofthe hGM-CSF receptor.
 27. The method of claim 25, wherein theanti-hGM-CSF antibody is a polyclonal antibody.
 28. The method of claim25, wherein the anti-hGM-CSF antibody is a monoclonal antibody.
 29. Themethod of claim 25, wherein the anti-hGM-CSF antibody is an antibodyfragment that is a Fab, a Fab′, a F(ab′)2, a scFv, or a dAB.
 30. Themethod of claim 29, wherein the antibody fragment is conjugated topolyethylene glycol.
 31. The method of claim 25, wherein theanti-hGM-CSF antibody has an affinity ranging from about 5 pM to about50 pM.
 32. The method of claim 25, wherein the anti-hGM-CSF antibody isa neutralizing antibody.
 33. The method of claim 25, wherein theanti-hGM-CSF antibody is a recombinant or chimeric antibody.
 34. Themethod of claim 25, wherein the anti-hGM-CSF antibody is a humanantibody.
 35. The method of claim 25, wherein the anti-hGM-CSF antibodycomprises a human variable region.
 36. The method of claim 25, whereinthe anti-hGM-CSF antibody comprises a human light chain constant region.37. The method of claim 25, wherein the anti-hGM-CSF antibody comprisesa human heavy chain constant region.
 38. The method of claim 37, whereinthe human heavy chain constant region is a gamma chain.
 39. The methodof claim 25, wherein the anti-hGM-CSF antibody binds to the same epitopeas chimeric 19/2.
 40. The method of claim 25, wherein the anti-hGM-CSFantibody comprises the VH region CDR3 and VL region CDR3 of chimeric19/2.
 41. The method of claim 25, wherein the anti-hGM-CSF antibodycomprises the VH region and VL region CDR1, CDR2, and CDR3 of chimeric19/2.
 42. The method of claim 25, wherein the anti-hGM-CSF antibodycomprises a VH region that comprises a CDR3 binding specificitydeterminant RQRFPY or RDRFPY, a J segment, and a V-segment, wherein theJ-segment comprises at least 95% identity to human JH4 (YFD YWGQGTLVTVSS) and the V-segment comprises at least 90% identity to a human germline VH1 1-02 or VH1 1-03 sequence; or a VH region that comprises a CDR3binding specificity determinant RQRFPY.
 43. The method of claim 42,wherein the J segment comprises YFDYWGQGTLVTVSS.
 44. The method of claim42, wherein the CDR3 comprises RQRFPYYFDY or RDRFPYYFDY.
 45. The methodof claim 42, wherein the VH region CDR1 is a human germline VH1 CDR1;the VH region CDR2 is a human germline VH1 CDR2; or both the CDR1 andCDR2 are from a human germline VH1 sequence.
 46. The method of claim 42,wherein the anti-hGM-CSF antibody comprises a VH CDR1, or a VH CDR2, orboth a VH CDR1 and a VH CDR2 as shown in a VH region set forth inFIG.
 1. 47. The method of claim 42, wherein the V-segment sequence has aVH V segment sequence shown in FIG.
 1. 48. The method of claim 42,wherein the VH has the sequence of VH#1, VH#2, VH#3, VH#4, or VH#5 setforth in FIG.
 1. 49. The method of claim 25, wherein the anti-hGM-CSFantibody comprises a VL-region that comprises a CDR3 comprising theamino acid sequence FNK or FNR.
 50. The method of claim 49, wherein theanti-hGM-CSF antibody comprises a human germline JK4 region.
 51. Themethod of claim 49, wherein the VL region CDR3 comprises QQFN(K/R)SPLT.52. The method of claim 51, wherein the anti-hGM-CSF antibody comprisesa VL region that comprises a CDR3 comprising QQFNKSPLT.
 53. The methodof claim 49, where the VL region comprises a CDR1, or a CDR2, or both aCDR1 and CDR2 of a VL region shown in FIG.
 1. 54. The method of claim49, wherein the VL region comprises a V segment that has at least 95%identity to the VKIII A27 V-segment sequence as shown in FIG.
 1. 55. Themethod of claim 49, wherein the VL region has the sequence of VK#1,VK#2, VK#3, or VK#4 set forth in FIG.
 1. 56. The method of claim 25,wherein the anti-hGM-CSF antibody has a VH region CDR3 bindingspecificity determinant RQRFPY or RDRFPY and a VL region that has a CDR3comprising QQFNKSPLT.
 57. The method of claim 25, wherein theanti-hGM-CSF antibody has a VH region sequence set forth in FIG. 1 and aVL region sequence set forth in FIG.
 1. 58. The method of claim 25,wherein the VH region or the VL region, or both the VH and VL regionamino acid sequences comprise a methionine at the N-terminus.
 59. Themethod of claim 1, wherein the hGM-CSF antagonist is selected from thegroup comprising of an anti-hGM-CSF receptor antibody or a solublehGM-CSF receptor, a cytochrome b562 antibody mimetic, a hGM-CSF peptideanalog, an adnectin, a lipocalin scaffold antibody mimetic, a calixareneantibody mimetic, and an antibody like binding peptidomimetic.
 60. AhGM-CSF antagonist for use in a method of inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity in asubject, the method comprising a step of administering a recombinanthGM-CSF antagonist to the subject, wherein said administering inhibitsor reduces the incidence of immunotherapy-related toxicity in saidsubject.
 61. The hGM-CSF antagonist of claim 60, wherein saidimmunotherapy comprises adoptive cell transfer, administration ofmonoclonal antibodies, administration of cytokines, administration of acancer vaccine, T cell engaging therapies, or any combination thereof.62. The hGM-CSF antagonist of claim 61, wherein said adoptive celltransfer comprises administering chimeric antigen receptor-expressingT-cells (CAR T-cells), T-cell receptor (TCR) modified T-cells,tumor-infiltrating lymphocytes (TIL), chimeric antigen receptor(CAR)-modified natural killer cells, or dendritic cells, or anycombination thereof.
 63. The hGM-CSF antagonist of claim 61, whereinsaid monoclonal antibody is selected from a group comprising: anti-CD3,anti-CD52, anti-PD1, anti-PD-L1, anti-CTLA4, anti-CD20, anti-BCMAantibodies, bi-specific antibodies, or bispecific T-cell engager (BiTE)antibodies, or any combination thereof.
 64. The hGM-CSF antagonist ofclaim 61, wherein said cytokines are selected from a group comprising:IFNα, IFNβ, IFNγ, IFNλ, IL-2, IL-7, IL-15, IL-21, IL-11, IL-12, IL-18,hGM-CSF, TNFα, or any combination thereof.
 65. The hGM-CSF antagonist ofclaim 60, wherein said inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises reducing theconcentration of at least one inflammation-associated factor in theserum or in the CSF of said subject is decreased.
 66. The hGM-CSFantagonist of claim 65, wherein said inflammation-associated factor isselected from a group comprising: C-reactive protein, GM-CSF, IL-2,sIL2Rα, IL-5, IL-6, IL-8, IL-10, IP10, IL-15, MCP-1, MIG, MIP1β, IFNγ,CX3CR1, or TNFα, or any combination thereof.
 67. The hGM-CSF antagonistof claim 60, wherein administration of said recombinant GM-CSFantagonist does not reduce the efficacy of said immunotherapy.
 68. ThehGM-CSF antagonist of claim 60, wherein said immunotherapy isadministered at higher doses that it would be administered without theadministration of said hGM-CSF antagonists.
 69. The hGM-CSF antagonistof claim 60, wherein administration of said recombinant hGM-CSFantagonist occurs prior to, concurrent with, or following saidimmunotherapy.
 70. The hGM-CSF antagonist of claim 60, wherein saidrecombinant hGM-CSF antagonist is co-administered with corticosteroids,anti-IL-6 antibodies, tocilizumab, cyclosporine, antiepileptics,benzodiazepines, acetazolamide, hyperventilation therapy, orhyperosmolar therapy, or any combination thereof.
 71. The hGM-CSFantagonist of claim 60, wherein said immunotherapy-related toxicitycomprises a brain disease, damage or malfunction.
 72. The hGM-CSFantagonist of claim 71, wherein said brain disease, damage ormalfunction comprises CAR-T cell related neurotoxicity or CAR-T cellrelated encephalopathy syndrome (CRES).
 73. The hGM-CSF antagonist ofclaim 72, wherein the CAR-T cell related neurotoxicity is reduced byabout 90% compared to a reduction in neurotoxicity in a subject treatedwith CAR-T cells and a control antibody.
 74. The hGM-CSF antagonist ofclaim 71, wherein said inhibiting or reducing incidence of a braindisease, damage or malfunction comprises reducing headaches, delirium,anxiety, tremor, seizure activity, confusion, alterations inwakefulness, hallucinations, dysphasia, ataxia, apraxia, facial nervepalsy, motor weakness, seizures, nonconvulsive EEG seizures, coma,endothelial activation, vascular leak, intravascular coagulation, or anycombination thereof in said subject.
 75. The hGM-CSF antagonist of claim71, wherein the serum concentration of ANG2 or VWF, or the serumANG2:ANG1 ratio of said subject is reduced.
 76. The hGM-CSF antagonistof claim 71, wherein said subject has a body temperature above 38° C.,IL6 serum concentration above 16 pg/ml, or MCP1 serum concentrationabove 1,300 pg/ml during the first 36 hours after infusion of said CAR-Tcells.
 77. The hGM-CSF antagonist of claim 70, wherein said subject ispredisposed to have said brain disease, damage or malfunction.
 78. ThehGM-CSF antagonist of claim 70, wherein said subject has an ANG2:ANG1ratio in serum above 1 prior to the infusion of said CAR-T cells. 79.The hGM-CSF antagonist of claim 60, wherein said immunotherapy-relatedtoxicity comprises hemophagocytic lymphohistiocytosis (HLH) ormacrophage-activation syndrome (MAS).
 80. The hGM-CSF antagonist ofclaim 79, wherein said inhibiting or reducing incidence of HLH or MAScomprises increasing survival time and/or time to relapse, reducingmacrophage activation, reducing T cell activation, reducing theconcentration of circulating IFNγ, or reducing the concentration ofcirculating of hGM-CSF, or any combination thereof.
 81. The hGM-CSFantagonist of claim 79, wherein said subject presents with fever,splenomegaly, cytopenias involving two or more lines,hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis, low orabsent NK-cell activity, ferritin serum concentration above 500 U/ml, orsoluble CD25 serum concentration above 2400 U/ml, or any combinationthereof.
 82. The hGM-CSF antagonist of claim 79, wherein said subject ispredisposed to acquiring HLH or MAS.
 83. The hGM-CSF antagonist of claim77, wherein said subject carries a mutation in a gene selected from:PRF1, UNC13D, STX11, STXBP2, or RAB27A, or has reduced expression ofperforin, or any combination thereof.
 84. The hGM-CSF antagonist ofclaim 60, wherein the hGM-CSF antagonist is an anti-hGM-CSF antibody.85. The hGM-CSF antagonist of claim 84, wherein the anti-hGM-CSFantibody blocks binding of hGM-CSF to the alpha subunit of the hGM-CSFreceptor.
 86. The hGM-CSF antagonist of claim 84, wherein theanti-hGM-CSF antibody is a polyclonal antibody.
 87. The hGM-CSFantagonist of claim 84, wherein the anti-hGM-CSF antibody is amonoclonal antibody.
 88. The hGM-CSF antagonist of claim 84, wherein theanti-hGM-CSF antibody is an antibody fragment that is a Fab, a Fab′, aF(ab′)2, a scFv, or a dAB.
 89. The hGM-CSF antagonist of claim 88,wherein the antibody fragment is conjugated to polyethylene glycol. 90.The hGM-CSF antagonist of claim 84, wherein the anti-hGM-CSF antibodyhas an affinity ranging from about 5 pM to about 50 pM.
 91. The hGM-CSFantagonist of claim 84, wherein the anti-hGM-CSF antibody is aneutralizing antibody.
 92. The hGM-CSF antagonist of claim 84, whereinthe anti-hGM-CSF antibody is a recombinant or chimeric antibody.
 93. ThehGM-CSF antagonist of claim 84, wherein the anti-hGM-CSF antibody is ahuman antibody.
 94. The hGM-CSF antagonist of claim 84, wherein theanti-hGM-CSF antibody comprises a human variable region.
 95. The hGM-CSFantagonist of claim 84, wherein the anti-hGM-CSF antibody comprises ahuman light chain constant region.
 96. The hGM-CSF antagonist of claim84, wherein the anti-hGM-CSF antibody comprises a human heavy chainconstant region.
 97. The hGM-CSF antagonist of claim 96, wherein thehuman heavy chain constant region is a gamma chain.
 98. The hGM-CSFantagonist of claim 84, wherein the anti-hGM-CSF antibody binds to thesame epitope as chimeric 19/2.
 99. The hGM-CSF antagonist of claim 84,wherein the anti-hGM-CSF antibody comprises the VH region CDR3 and VLregion CDR3 of chimeric 19/2.
 100. The hGM-CSF antagonist of claim 84,wherein the anti-hGM-CSF antibody comprises the VH region and VL regionCDR1, CDR2, and CDR3 of chimeric 19/2.
 101. The hGM-CSF antagonist ofclaim 84, wherein the anti-hGM-CSF antibody comprises a VH region thatcomprises a CDR3 binding specificity determinant RQRFPY or RDRFPY, a Jsegment, and a V-segment, wherein the J-segment comprises at least 95%identity to human JH4 (YFD YWGQGTL VTVSS) and the V-segment comprises atleast 90% identity to a human germ line VH1 1-02 or VH1 1-03 sequence;or a VH region that comprises a CDR3 binding specificity determinantRQRFPY.
 102. The hGM-CSF antagonist of claim 101, wherein the J segmentcomprises YFDYWGQGTLVTVSS.
 103. The hGM-CSF antagonist of claim 101,wherein the CDR3 comprises RQRFPYYFDY or RDRFPYYFDY.
 104. The hGM-CSFantagonist of claim 101, wherein the VH region CDR1 is a human germlineVH1 CDR1; the VH region CDR2 is a human germline VH1 CDR2; or both theCDR1 and CDR2 are from a human germline VH1 sequence.
 105. The hGM-CSFantagonist of claim 101, wherein the anti-hGM-CSF antibody comprises aVH CDR1, or a VH CDR2, or both a VH CDR1 and a VH CDR2 as shown in a VHregion set forth in FIG.
 1. 106. The hGM-CSF antagonist of claim 101,wherein the V-segment sequence has a VH V segment sequence shown inFIG.
 1. 107. The hGM-CSF antagonist of claim 101, wherein the VH has thesequence of VH#1, VH#2, VH#3, VH#4, or VH#5 set forth in FIG.
 1. 108.The GM-CSF antagonist of claim 84, wherein the anti-hGM-CSF antibodycomprises a VL-region that comprises a CDR3 comprising the amino acidsequence FNK or FNR.
 109. The hGM-CSF antagonist of claim 108, whereinthe anti-hGM-CSF antibody comprises a human germline JK4 region. 110.The hGM-CSF antagonist of claim 108, wherein the VL region CDR3comprises QQFN(K/R)SPLT.
 111. The hGM-CSF antagonist of claim 110,wherein the anti-hGM-CSF antibody comprises a VL region that comprises aCDR3 comprising QQFNKSPLT.
 112. The hGM-CSF antagonist of claim 108,where the VL region comprises a CDR1, or a CDR2, or both a CDR1 and CDR2of a VL region shown in FIG.
 1. 113. The hGM-CSF antagonist of claim108, wherein the VL region comprises a V segment that has at least 95%identity to the VKIII A27 V-segment sequence as shown in FIG.
 1. 114.The GM-CSF antagonist of claim 108, wherein the VL region has thesequence of VK#1, VK#2, VK#3, or VK#4 set forth in FIG.
 1. 115. ThehGM-CSF antagonist of claim 84, wherein the anti-hGM-CSF antibody has aVH region CDR3 binding specificity determinant RQRFPY or RDRFPY and a VLregion that has a CDR3 comprising QQFNKSPLT.
 116. The hGM-CSF antagonistof claim 84, wherein the anti-hGM-CSF antibody has a VH region sequenceset forth in FIG. 1 and a VL region sequence set forth in FIG.
 1. 117.The hGM-CSF antagonist of claim 84, wherein the VH region or the VLregion, or both the VH and VL region amino acid sequences comprise amethionine at the N-terminus.
 118. The hGM-CSF antagonist of claim 60,wherein the hGM-CSF antagonist is selected from the group comprising ofan anti-hGM-CSF receptor antibody, a soluble hGM-CSF receptor, acytochrome b562 antibody mimetic, a hGM-CSF peptide analog, an adnectin,a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic,and an antibody like binding peptidomimetic.
 119. A method of increasingthe efficacy of CAR-T immunotherapy in a subject, the method comprisinga step of administering a recombinant hGM-CSF antagonist to the subject,wherein said administering increases the efficacy of CAR-T immunotherapyin said subject.
 120. The method of claim 119, wherein saidadministering a recombinant hGM-CSF antagonist occurs prior to,concurrent with, or following said CAR-T immunotherapy.
 121. The methodof claim 120, wherein said increased efficacy comprises increased CAR-Tcell expansion, reduced myeloid-derived suppressor cells (MDSC) thatinhibit T-cell function, synergy with a checkpoint inhibitor, or anycombination thereof.
 122. The method of claim 121, wherein saidincreased CAR-T cell expansion comprises at least a 50% increasecompared to a control.
 123. The method of claim 121, wherein saidincreased CAR-T cell expansion comprises at least a one quarter logexpansion compared to a control.
 124. The method of claim 121, whereinsaid increased cell expansion comprises at least a one half logexpansion compared to a control.
 125. The method of claim 121, whereinsaid increased cell expansion comprises at least a one log expansioncompared to a control.
 126. The method of claim 121, wherein saidincreased cell expansion comprises a greater than one log expansioncompared to a control.
 127. The method of claim 119, wherein the hGM-CSFantagonist comprises a neutralizing antibody.
 128. The method of claim127, wherein the neutralizing antibody is a monoclonal antibody.
 129. Amethod of inhibiting or reducing the incidence or the severity of CAR-Trelated toxicity in a subject, the method comprising a step ofadministering a recombinant hGM-CSF antagonist to the subject, whereinsaid administering inhibits or reduces the incidence or the severity ofCAR-T related toxicity in said subject.
 130. The method of claim 129,wherein said CAR-T related toxicity comprises neurotoxicity, CRS, or acombination thereof.
 131. The method of claim 130, wherein the CAR-Tcell related neurotoxicity is reduced by about 50% compared to areduction in neurotoxicity in a subject treated with CAR-T cells and acontrol antibody.
 132. The method of claim 129, wherein said inhibitingor reducing incidence of CRS comprises increasing survival time and/ortime to relapse, reducing macrophage activation, reducing T cellactivation, or reducing the concentration of circulating hGM-CSF, or anycombination thereof.
 133. The method of claim 129, wherein said subjectpresents with fever (with or without rigors, malaise, fatigue, anorexia,myalgia, arthralgia, nausea, vomiting, headache, skin rash, diarrhea,tachypnea, hypoxemia, hypoxia, shock, cardiovascular tachycardia,widened pulse pressure, hypotension, capillary leak, increased earlycardiac output, diminished late cardiac output, elevated D-dimer,hypofibrinogenemia with or without bleeding, azotemia, transaminitis,hyperbilirubinemia, mental status changes, confusion, delirium, frankaphasia, hallucinations, tremor, dysmetria, altered gait, seizures,organ failure, or any combination thereof.
 134. The method of claim 129,wherein the inhibiting or reducing the incidence or the severity ofCAR-T related toxicity comprises preventing the onset of CAR-T relatedtoxicity.
 135. A method of blocking or reducing hGM-CSF expression in acell, comprising knocking out or silencing hGM-CSF gene expression inthe cell.
 136. The method of claim 135, wherein the blocking or reducinghGM-CSF expression comprises siRNA, CRISPR, RNAi, ddRNAi or TALENs. 137.The method of claim 1, wherein the subject is a human.
 138. Apharmaceutical composition comprising an anti-hGM-CSF antagonist ofclaim 60.